Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/US2010/055643 filed on Nov. 5, 2010 and claims priority from U.S.provisional application Ser. No. 61/310,430 filed on Mar. 4, 2010 andU.S. provisional application Ser. No. 61/258,999 filed on Nov. 6, 2009,which applications are fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to a deposit of biologicalmaterial, which deposit is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to polypeptides having cellobiohydrolaseactivity and polynucleotides encoding the polypeptides. The inventionalso relates to nucleic acid constructs, vectors, and host cellscomprising the polynucleotides as well as methods of producing and usingthe polypeptides.

2. Description of the Related Art

Cellulose is a polymer of the simple sugar glucose linked by beta-1,4bonds. Many microorganisms produce enzymes that hydrolyze beta-linkedglucans. These enzymes include endoglucanases, cellobiohydrolases, andbeta-glucosidases. Endoglucanases digest the cellulose polymer at randomlocations, opening it to attack by cellobiohydrolases.Cellobiohydrolases sequentially release molecules of cellobiose from theends of the cellulose polymer. Cellobiose is a water-solublebeta-1,4-linked dimer of glucose. Beta-glucosidases hydrolyze cellobioseto glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the cellulose is converted to glucose,the glucose is easily fermented by yeast into ethanol.

It would be advantageous in the art to improve the ability toenzymatically degrade lignocellulosic feedstocks.

The present invention provides polypeptides having cellobiohydrolaseactivity and polynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingcellobiohydrolase activity selected from the group consisting of:

(a) a polypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 2;

(b) a polypeptide encoded by a polynucleotide that hybridizes undermedium stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 1, or (iii) the full-lengthcomplementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide having at least 65%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the genomic DNA sequence thereof;

(d) a variant comprising a substitution, deletion, and/or insertion ofone or more (several) amino acids of the mature polypeptide of SEQ IDNO: 2; and

(e) a fragment of the polypeptide of (a), (b), (c), or (d) that hascellobiohydrolase activity.

The present invention also relates to isolated polynucleotides encodingthe polypeptides of the present invention; nucleic acid constructs,recombinant expression vectors, and recombinant host cells comprisingthe polynucleotides; and methods of producing the polypeptides.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellobiohydrolase activity of the present invention. In apreferred aspect, the method further comprises recovering the degradedor converted cellulosic material.

The present invention also relates to methods of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellobiohydrolase activity of the present invention; (b)fermenting the saccharified cellulosic material with one or more(several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of apolypeptide having cellobiohydrolase activity of the present invention.In a preferred aspect, the fermenting of the cellulosic materialproduces a fermentation product. In one aspect, the method furthercomprises recovering the fermentation product from the fermentation.

The present invention also relates to a polynucleotide encoding a signalpeptide comprising or consisting of amino acids 1 to 16 of SEQ ID NO: 2,which is operably linked to a gene encoding a protein; nucleic acidconstructs, expression vectors, and recombinant host cells comprisingthe polynucleotides; and methods of producing a protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect on hydrolysis of milled unwashed PCS byhigh-temperature enzyme compositions with and without Trichophaeasaccata Family GH6 polypeptide at 50-65° C.

DEFINITIONS

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91), which catalyzes thehydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellooligosaccharides, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing ends ofthe chain (Teeri, 1997, Crystalline cellulose degradation: New insightinto the function of cellobiohydrolases, Trends in Biotechnology 15:160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: whyso efficient on crystalline cellulose?, Biochem. Soc. Trans. 26:173-178). For purposes of the present invention, cellobiohydrolaseactivity is determined according to the procedures described by Lever etal., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBSLetters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters,187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. Inthe present invention, the Lever et al. method can be employed to assesshydrolysis of cellulose in corn stover, while the methods of vanTilbeurgh et al. and Tomme et al. can be used to determine thecellobiohydrolase activity on a fluorescent disaccharide derivative,4-methylumbelliferyl-β-D-lactoside.

The polypeptides of the present invention have at least 20%, e.g., atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, and at least 100% of the cellobiohydrolaseactivity of the mature polypeptide of SEQ ID NO: 2.

Family 6 glycoside hydrolase: The term “Family 6 glycoside hydrolase” or“Family GH6” or “GH6” means a polypeptide falling into the glycosidehydrolase Family 6 according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman No 1filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No 1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-20mg of cellulolytic enzyme protein/g of cellulose in PCS for 3-7 days at50° C. compared to a control hydrolysis without addition of cellulolyticenzyme protein. Typical conditions are 1 ml reactions, washed orunwashed PCS, 5% insoluble solids, 50 mM sodium acetate pH 5, 1 mMMnSO₄, 50° C., 72 hours, sugar analysis by AMINEX® HPX-87H column(Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Endoglucanase: The term “endoglucanase” means an endo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4), which catalysesendohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose, cellulosederivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such ascereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21), which catalyzes the hydrolysis ofterminal non-reducing beta-D-glucose residues with the release ofbeta-D-glucose. For purposes of the present invention, beta-glucosidaseactivity is determined according to the basic procedure described byVenturi et al., 2002, Extracellular beta-D-glucosidase from Chaetomiumthermophilum var. coprophilum: production, purification and somebiochemical properties, J. Basic Microbiol. 42: 55-66. One unit ofbeta-glucosidase is defined as 1.0 μmole of p-nitrophenolate anionproduced per minute at 25° C., pH 4.8 from 1 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodiumcitrate containing 0.01% TWEEN® 20.

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide that catalyzes the enhancement of the hydrolysis of acellulosic material by enzyme having cellulolytic activity. For purposesof the present invention, cellulolytic enhancing activity is determinedby measuring the increase in reducing sugars or the increase of thetotal of cellobiose and glucose from the hydrolysis of a cellulosicmaterial by cellulolytic enzyme under the following conditions: 1-50 mgof total protein/g of cellulose in PCS, wherein total protein iscomprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/wprotein of a GH61 polypeptide having cellulolytic enhancing activity for1-7 days at 50° C. compared to a control hydrolysis with equal totalprotein loading without cellulolytic enhancing activity (1-50 mg ofcellulolytic protein/g of cellulose in PCS). In a preferred aspect, amixture of CELLUCLAST® 1.5 L (Novozymes A/S, Bagsyrd, Denmark) in thepresence of 2-3% of total protein weight Aspergillus oryzaebeta-glucosidase (recombinantly produced in Aspergillus oryzae accordingto WO 02/095014) or 2-3% of total protein weight Aspergillus fumigatusbeta-glucosidase (recombinantly produced in Aspergillus oryzae asdescribed in WO 2002/095014) of cellulase protein loading is used as thesource of the cellulolytic activity.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, more preferably at least 1.05-fold, more preferably at least1.10-fold, more preferably at least 1.25-fold, more preferably at least1.5-fold, more preferably at least 2-fold, more preferably at least3-fold, more preferably at least 4-fold, more preferably at least5-fold, even more preferably at least 10-fold, and most preferably atleast 20-fold.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (several) enzymes thathydrolyze a hemicellulosic material. See, for example, Shallom, D. andShoham, Y. Microbial hemicellulases. Current Opinion In Microbiology,2003, 6(3): 219-228). Hemicellulases are key components in thedegradation of plant biomass. Examples of hemicellulases include, butare not limited to, an acetylmannan esterase, an acetyxylan esterase, anarabinanase, an arabinofuranosidase, a coumaric acid esterase, aferuloyl esterase, a galactosidase, a glucuronidase, a glucuronoylesterase, a mannanase, a mannosidase, a xylanase, and a xylosidase. Thesubstrates of these enzymes, the hemicelluloses, are a heterogeneousgroup of branched and linear polysaccharides that are bound via hydrogenbonds to the cellulose microfibrils in the plant cell wall, crosslinkingthem into a robust network. Hemicelluloses are also covalently attachedto lignin, forming together with cellulose a highly complex structure.The variable structure and organization of hemicelluloses require theconcerted action of many enzymes for its complete degradation. Thecatalytic modules of hemicellulases are either glycoside hydrolases(GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs),which hydrolyze ester linkages of acetate or ferulic acid side groups.These catalytic modules, based on homology of their primary sequence,can be assigned into GH and CE families marked by numbers. Somefamilies, with overall similar fold, can be further grouped into clans,marked alphabetically (e.g., GH-A). A most informative and updatedclassification of these and other carbohydrate active enzymes isavailable on the Carbohydrate-Active Enzymes (CAZy) database.Hemicellulolytic enzyme activities can be measured according to Ghoseand Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, Recent progress in the assays of xylanolytic enzymes, 2006,Journal of the Science of Food and Agriculture 86(11): 1636-1647;Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrateesterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek,1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctionalbeta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. The most common total xylanolytic activity assay is basedon production of reducing sugars from polymeric 4-O-methylglucuronoxylan as described in Bailey, Biely, Poutanen, 1992,Interlaboratory testing of methods for assay of xylanase activity,Journal of Biotechnology 23(3): 257-270. Xylanase activity can also bedetermined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TritonX-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unit ofxylanase activity is defined as 1.0 μmole of azurine produced per minuteat 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mMsodium phosphate pH 6 buffer.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%Triton X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unitof xylanase activity is defined as 1.0 μmole of azurine produced perminute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6 buffer.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta-(4)-xylooligosaccharides, to remove successive D-xylose residuesfrom the non-reducing termini. For purposes of the present invention,one unit of beta-xylosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20.

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyses the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20. One unit of acetylxylan esterase is definedas the amount of enzyme capable of releasing 1 μmole of p-nitrophenolateanion per minute at pH 5, 25° C.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl(feruloyl)group from an esterified sugar, which is usually arabinose in “natural”substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloylesterase is also known as ferulic acid esterase, hydroxycinnamoylesterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, orFAE-II. For purposes of the present invention, feruloyl esteraseactivity is determined using 0.5 mM p-nitrophenylferulate as substratein 50 mM sodium acetate pH 5.0. One unit of feruloyl esterase equals theamount of enzyme capable of releasing 1 μmole of p-nitrophenolate anionper minute at pH 5, 25° C.

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Cellulosic material: The cellulosic material can be any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, herbaceous material,agricultural residue, forestry residue, municipal solid waste, wastepaper, and pulp and paper mill residue (see, for example, Wiselogel etal., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp.105-118, Taylor & Francis, Washington D.C.; Wyman, 1994, BioresourceTechnology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion ofLignocellulosics, in Advances in Biochemical Engineering/Biotechnology,T. Scheper, managing editor, Volume 65, pp. 23-40, Springer-Verlag, NewYork). It is understood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is lignocellulose.

In one aspect, the cellulosic material is herbaceous material. Inanother aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is forestry residue. In anotheraspect, the cellulosic material is municipal solid waste. In anotheraspect, the cellulosic material is waste paper. In another aspect, thecellulosic material is pulp and paper mill residue.

In another aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is corn fiber. In another aspect, thecellulosic material is corn cob. In another aspect, the cellulosicmaterial is orange peel. In another aspect, the cellulosic material isrice straw. In another aspect, the cellulosic material is wheat straw.In another aspect, the cellulosic material is switch grass. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is bagasse.

In another aspect, the cellulosic material is microcrystallinecellulose. In another aspect, the cellulosic material is bacterialcellulose. In another aspect, the cellulosic material is algalcellulose. In another aspect, the cellulosic material is cotton linter.In another aspect, the cellulosic material is amorphous phosphoric-acidtreated cellulose. In another aspect, the cellulosic material is filterpaper.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid.

Xylan-containing material: The term “xylan-containing material” isdefined herein as any material comprising a plant cell wallpolysaccharide containing a backbone of beta-(1-4)-linked xyloseresidues. Xylans of terrestrial plants are heteropolymers possessing abeta-(1-4)-D-xylopyranose backbone, which is branched by shortcarbohydrate chains. They comprise D-glucuronic acid or its 4-O-methylether, L-arabinose, and/or various oligosaccharides, composed ofD-xylose, L-arabinose, D- or L-galactose, and D-glucose. Xylan-typepolysaccharides can be divided into homoxylans and heteroxylans, whichinclude glucuronoxylans, (arabino)glucuronoxylans,(glucurono)arabinoxylans, arabinoxylans, and complex heteroxylans. See,for example, Ebringerova et al., 2005, Adv. Polym. Sci. 186: 1-67.

In the methods of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Isolated or purified: The term “isolated” or “purified” means apolypeptide or polynucleotide that is removed from at least onecomponent with which it is naturally associated. For example, apolypeptide may be at least 1% pure, e.g., at least 5% pure, at least10% pure, at least 20% pure, at least 40% pure, at least 60% pure, atleast 80% pure, at least 90% pure, or at least 95% pure, as determinedby SDS-PAGE, and a polynucleotide may be at least 1% pure, e.g., atleast 5% pure, at least 10% pure, at least 20% pure, at least 40% pure,at least 60% pure, at least 80% pure, at least 90% pure, or at least 95%pure, as determined by agarose electrophoresis.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 17 to 447 of SEQ ID NO: 2 based on theSignalP program (Nielsen et al., 1997, Protein Engineering 10:1-6) thatpredicts amino acids 1 to 16 of SEQ ID NO: 2 are a signal peptide. It isknown in the art that a host cell may produce a mixture of two of moredifferent mature polypeptides (i.e., with a different C-terminal and/orN-terminal amino acid) expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving cellobiohydrolase activity. In one aspect, the mature polypeptidecoding sequence is nucleotides 109 to 1401 of SEQ ID NO: 1 based on theSignalP program (Nielsen et al., 1997, supra) that predicts nucleotides61 to 108 of SEQ ID NO: 1 encode a signal peptide. In another aspect,the mature polypeptide coding sequence is the genomic DNA sequence ofnucleotides 109 to 1401 of SEQ ID NO: 1.

Sequence Identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the −nobrief option) is used as the percent identity andis calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the −nobriefoption) is used as the percent identity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Fragment: The term “fragment” means a polypeptide having one or more(several) amino acids deleted from the amino and/or carboxyl terminus ofa mature polypeptide; wherein the fragment has cellobiohydrolaseactivity. In one aspect, a fragment contains at least 390 amino acidresidues, e.g., at least 410 amino acid residues or at least 430 aminoacid residues.

Subsequence: The term “subsequence” means a polynucleotide having one ormore (several) nucleotides deleted from the 5′ and/or 3′ end of a maturepolypeptide coding sequence; wherein the subsequence encodes a fragmenthaving cellobiohydrolase activity. In one aspect, a subsequence containsat least 1170 nucleotides, e.g., at least 1230 nucleotides or at least1290 nucleotides.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which usually begins with the ATG start codon oralternative start codons such as GTG and TTG and ends with a stop codonsuch as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA,synthetic, or recombinant polynucleotide.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic cell. cDNA lacks intron sequences that may be presentin the corresponding genomic DNA. The initial, primary RNA transcript isa precursor to mRNA that is processed through a series of steps,including splicing, before appearing as mature spliced mRNA.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic. The term nucleic acid construct issynonymous with the term “expression cassette” when the nucleic acidconstruct contains the control sequences required for expression of acoding sequence of the present invention.

Control sequences: The term “control sequences” means all componentsnecessary for the expression of a polynucleotide encoding a polypeptideof the present invention. Each control sequence may be native or foreignto the polynucleotide encoding the polypeptide or native or foreign toeach other. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs the expression of the coding sequence.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to additional nucleotides thatprovide for its expression.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, and the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Variant: The term “variant” means a polypeptide having cellobiohydrolaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion of one or more (several) amino acid residues at one ormore (several) positions. A substitution means a replacement of an aminoacid occupying a position with a different amino acid; a deletion meansremoval of an amino acid occupying a position; and an insertion meansadding one or more (several) amino acids, e.g., 1-5 amino acids,adjacent to an amino acid occupying a position.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides HavingCellobiohydrolase Activity

The present invention relates to isolated polypeptides havingcellobiohydrolase activity selected from the group consisting of:

(a) a polypeptide having at least 65% sequence identity to the maturepolypeptide of SEQ ID NO: 2;

(b) a polypeptide encoded by a polynucleotide that hybridizes undermedium stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 1, or (iii) the full-lengthcomplementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide having at least 65%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the genomic DNA sequence thereof;

(d) a variant comprising a substitution, deletion, and/or insertion ofone or more (several) amino acids of the mature polypeptide of SEQ IDNO: 2; and

(f) a fragment of a polypeptide of (a), (b), (c), or (d) that hascellobiohydrolase activity.

The present invention relates to isolated polypeptides having a sequenceidentity to the mature polypeptide of SEQ ID NO: 2 of at least 65%,e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%,which have cellobiohydrolase activity. In one aspect, the polypeptidesdiffer by no more than ten amino acids, e.g., by five amino acids, byfour amino acids, by three amino acids, by two amino acids, and by oneamino acid from the mature polypeptide of SEQ ID NO: 2.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 2 or an allelic variantthereof; or is a fragment thereof having cellobiohydrolase activity. Inanother aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, thepolypeptide comprises or consists of amino acids 17 to 447 of SEQ ID NO:2.

The present invention also relates to isolated polypeptides havingcellobiohydrolase activity that are encoded by polynucleotides thathybridize under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) the full-length complementary strandof (i) or (ii) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe amino acid sequence of SEQ ID NO: 2 or a fragment thereof, may beused to design nucleic acid probes to identify and clone DNA encodingpolypeptides having cellobiohydrolase activity from strains of differentgenera or species according to methods well known in the art. Inparticular, such probes can be used for hybridization with the genomicDNA or cDNA of the genus or species of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 14, e.g., at least 25, atleast 35, or at least 70 nucleotides in length. Preferably, the nucleicacid probe is at least 100 nucleotides in length, e.g., at least 200nucleotides, at least 300 nucleotides, at least 400 nucleotides, atleast 500 nucleotides, at least 600 nucleotides, at least 700nucleotides, at least 800 nucleotides, or at least 900 nucleotides inlength. Both DNA and RNA probes can be used. The probes are typicallylabeled for detecting the corresponding gene (for example, with ³²P, ³H,³⁵S, biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having cellobiohydrolase activity. Genomic orother DNA from such other strains may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that is homologous with SEQ ID NO: 1 ora subsequence thereof, the carrier material is preferably used in aSouthern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto SEQ ID NO: 1; the mature polypeptide coding sequence of SEQ ID NO: 1;or the genomic DNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 1; its full-length complementary strand; or a subsequencethereof; under very low to very high stringency conditions. Molecules towhich the nucleic acid probe hybridizes under these conditions can bedetected using, for example, X-ray film.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1 or the genomic DNA sequence thereof. In anotheraspect, the nucleic acid probe is a polynucleotide that encodes thepolypeptide of SEQ ID NO: 2 or the mature polypeptide thereof; or afragment thereof. In another preferred aspect, the nucleic acid probe isSEQ ID NO: 1 or the genomic DNA sequence thereof. In another aspect, thenucleic acid probe is the polynucleotide contained in plasmid pMStr199which is contained in E. coli DSM 23379, wherein the polynucleotideencodes a polypeptide having cellobiohydrolase activity. In anotheraspect, the nucleic acid probe is the mature polypeptide coding regioncontained in plasmid pMStr199 which is contained in E. coli DSM 23379.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and either 25% formamide for very lowand low stringencies, 35% formamide for medium and medium-highstringencies, or 50% formamide for high and very high stringencies,following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed three times each for15 minutes using 2×SSC, 0.2% SDS at 45° C. (very low stringency), at 50°C. (low stringency), at 55° C. (medium stringency), at 60° C.(medium-high stringency), at 65° C. (high stringency), and at 70° C.(very high stringency).

For short probes of about 15 nucleotides to about 70 nucleotides inlength, stringency conditions are defined as prehybridization andhybridization at about 5° C. to about 10° C. below the calculated T_(m)using the calculation according to Bolton and McCarthy (1962, Proc.Natl. Acad. Sci. USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA perml following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed once in 6×SCC plus0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5°C. to 10° C. below the calculated T_(m).

The present invention also relates to isolated polypeptides havingcellobiohydrolase activity encoded by polynucleotides having a sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1 orthe genomic DNA sequence thereof of at least 65%, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%.

The present invention also relates to variants comprising asubstitution, deletion, and/or insertion of one or more (or several)amino acids of the mature polypeptide of SEQ ID NO: 2, or a homologoussequence thereof. Preferably, amino acid changes are of a minor nature,that is conservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for cellobiohydrolase activity toidentify amino acid residues that are critical to the activity of themolecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708.The active site of the enzyme or other biological interaction can alsobe determined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities ofessential amino acids can also be inferred from analysis of identitieswith polypeptides that are related to the parent polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 2 is not more than10, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9.

The polypeptide may be hybrid polypeptide in which a portion of onepolypeptide is fused at the N-terminus or the C-terminus of a portion ofanother polypeptide.

The polypeptide may be a fused polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusedpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator. Fusion proteins may also be constructedusing intein technology in which fusions are createdpost-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawsonet al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Cellobiohydrolase Activity

A polypeptide having cellobiohydrolase activity of the present inventionmay be obtained from microorganisms of any genus. For purposes of thepresent invention, the term “obtained from” as used herein in connectionwith a given source shall mean that the polypeptide encoded by apolynucleotide is produced by the source or by a strain in which thepolynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

The polypeptide may be a bacterial polypeptide. For example, thepolypeptide may be a gram-positive bacterial polypeptide such as aBacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, orStreptomyces polypeptide having cellobiohydrolase activity, or agram-negative bacterial polypeptide such as a Campylobacter, E. coli,Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria,Pseudomonas, Salmonella, or Ureaplasma polypeptide.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide.

In another aspect, the polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus polypeptide.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans polypeptide.

The polypeptide may also be a fungal polypeptide. For example, thepolypeptide may be a yeast polypeptide such as a Candida, Kluyveromyces,Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; ora filamentous fungal polypeptide such as an Acremonium, Agaricus,Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis,Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis,Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia,Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex,Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide.

In another aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulaturn, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechtysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa,Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide.

In another aspect, the polypeptide is an Trichophaea saccata polypeptidehaving cellobiohydrolase activity. In another aspect, the polypeptide isan Trichophaea saccata CBS 804.70 polypeptide having cellobiohydrolaseactivity, e.g., the polypeptide comprising the mature polypeptide of SEQID NO: 2.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) using the above-mentioned probes. Techniques for isolatingmicroorganisms from natural habitats are well known in the art. Thepolynucleotide encoding the polypeptide may then be obtained bysimilarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding apolypeptide has been detected with the probe(s), the polynucleotide canbe isolated or cloned by utilizing techniques that are well known tothose of ordinary skill in the art (see, e.g., Sambrook et al., 1989,supra).

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides from such genomic DNA can be effected, e.g., by usingthe well known polymerase chain reaction (PCR) or antibody screening ofexpression libraries to detect cloned DNA fragments with sharedstructural features. See, e.g., Innis et al., 1990, PCR: A Guide toMethods and Application, Academic Press, New York. Other nucleic acidamplification procedures such as ligase chain reaction (LCR), ligationactivated transcription (LAT) and polynucleotide-based amplification(NASBA) may be used. The polynucleotides may be cloned from a strain ofTrichophaea, or a related organism and thus, for example, may be anallelic or species variant of the polypeptide encoding region of thepolynucleotide.

The present invention also relates to isolated polynucleotidescomprising or consisting of polynucleotides having a degree of sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1 orthe genomic DNA sequence thereof of at least 65%, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, which encode apolypeptide having cellobiohydrolase activity.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., variantsthat differ in specific activity, thermostability, pH optimum, or thelike. The variant may be constructed on the basis of the polynucleotidepresented as the mature polypeptide coding sequence of SEQ ID NO: 1 orthe genomic DNA sequence thereof, e.g., a subsequence thereof, and/or byintroduction of nucleotide substitutions that do not result in a changein the amino acid sequence of the polypeptide, but which correspond tothe codon usage of the host organism intended for production of theenzyme, or by introduction of nucleotide substitutions that may giverise to a different amino acid sequence. For a general description ofnucleotide substitution, see, e.g., Ford et al., 1991, ProteinExpression and Purification 2: 95-107.

The present invention also relates to isolated polynucleotides encodingpolypeptides of the present invention, which hybridize under very lowstringency conditions, low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the genomic DNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 1, or(iii) the full-length complementary strand of (i) or (ii); or allelicvariants and subsequences thereof (Sambrook et al., 1989, supra), asdefined herein.

In one aspect, the polynucleotide comprises or consists of SEQ ID NO: 1,the mature polypeptide coding sequence of SEQ ID NO: 1, or the sequencecontained in plasmid pMStr199 which is contained in E. coli DSM 23379,or a subsequence of SEQ ID NO: 1 that encodes a fragment of SEQ ID NO: 2having cellobiohydrolase activity, such as the polynucleotide ofnucleotides 109 to 1401 of SEQ ID NO: 1.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or more(several) control sequences that direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide priorto its insertion into a vector may be desirable or necessary dependingon the expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter sequence, a polynucleotide thatis recognized by a host cell for expression of a polynucleotide encodinga polypeptide of the present invention. The promoter sequence containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, E. colilac operon, Streptomyces coelicolor agarase gene (dagA), and prokaryoticbeta-lactamase gene (VIIIa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci.USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983,Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are describedin “Useful proteins from recombinant bacteria” in Gilbert et al., 1980,Scientific American, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from a gene encoding a neutralalpha-amylase in Aspergilli in which the untranslated leader has beenreplaced by an untranslated leader from a gene encoding triose phosphateisomerase in Aspergilli; non-limiting examples include modifiedpromoters from the gene encoding neutral alpha-amylase in Aspergillusniger in which the untranslated leader has been replaced by anuntranslated leader from the gene encoding triose phosphate isomerase inAspergillus nidulans or Aspergillus oryzae); and mutant, truncated, andhybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, which is recognized by a host cell to terminate transcription.The terminator sequence is operably linked to the 3′-terminus of thepolynucleotide encoding the polypeptide. Any terminator that isfunctional in the host cell of choice may be used in the presentinvention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, whentranscribed is a nontranslated region of an mRNA that is important fortranslation by the host cell. The leader sequence is operably linked tothe 5′-terminus of the polynucleotide encoding the polypeptide. Anyleader sequence that is functional in the host cell of choice may beused.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular. Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. The foreign signal peptide coding sequence may be requiredwhere the coding sequence does not naturally contain a signal peptidecoding sequence. Alternatively, the foreign signal peptide codingsequence may simply replace the natural signal peptide coding sequencein order to enhance secretion of the polypeptide. However, any signalpeptide coding sequence that directs the expressed polypeptide into thesecretory pathway of a host cell of choice may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present at theN-terminus of a polypeptide, the propeptide sequence is positioned nextto the N-terminus of a polypeptide and the signal peptide sequence ispositioned next to the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the Aspergillus niger glucoamylasepromoter, Aspergillus oryzae TAKA alpha-amylase promoter, andAspergillus oryzae glucoamylase promoter may be used. Other examples ofregulatory sequences are those that allow for gene amplification. Ineukaryotic systems, these regulatory sequences include the dihydrofolatereductase gene that is amplified in the presence of methotrexate, andthe metallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more (several) convenientrestriction sites to allow for insertion or substitution of thepolynucleotide encoding the polypeptide at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the sequence into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more (several) selectable markersthat permit easy selection of transformed, transfected, transduced, orthe like cells. A selectable marker is a gene the product of whichprovides for biocide or viral resistance, resistance to heavy metals,prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or more(several) control sequences that direct the production of a polypeptideof the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any gram-positive or gram-negativebacterium. Gram-positive bacteria include, but not limited to, Bacillus,Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus,Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces.Gram-negative bacteria include, but not limited to, Campylobacter, E.coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter,Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Mol. Gen. Genet. 168: 111-115), by using competent cells (see, e.g.,Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), by electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or byconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may, forinstance, be effected by protoplast transformation (see, e.g., Hanahan,1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Doweret al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNAinto a Streptomyces cell may, for instance, be effected by protoplasttransformation and electroporation (see, e.g., Gong et al., 2004, FoliaMicrobiol. (Praha) 49: 399-405), by conjugation (see, e.g., Mazodier etal., 1989, J. Bacteriol. 171: 3583-3585), or by transduction (see, e.g.,Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). Theintroduction of DNA into a Pseudomonas cell may, for instance, beeffected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol.Methods 64: 391-397) or by conjugation (see, e.g., Pinedo and Smets,2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA intoa Streptococcus cell may, for instance, be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), by protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), by electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (asdefined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary ofThe Fungi, 8th edition, 1995, CAB International, University Press,Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al.,1995, supra, page 171) and all mitosporic fungi (Hawksworth et al.,1995, supra).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, F. A., Passmore, S. M., andDavenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9,1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In a preferred aspect, the cell is of the genus Trichophaea. In a morepreferred aspect, the cell is Trichophaea saccata. In a most preferredaspect, the cell is Trichophaea saccata CBS 804.70.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods well known in the art. Forexample, the cell may be cultivated by shake flask cultivation, andsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). If the polypeptide is secreted into thenutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it can be recovered fromcell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, centrifugation,filtration, extraction, spray-drying, evaporation, or precipitation.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989) to obtainsubstantially pure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising an isolatedpolynucleotide of the present invention so as to express and produce thepolypeptide in recoverable quantities. The polypeptide may be recoveredfrom the plant or plant part. Alternatively, the plant or plant partcontaining the polypeptide may be used as such for improving the qualityof a food or feed, e.g., improving nutritional value, palatability, andrheological properties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide may beconstructed in accordance with methods known in the art. In short, theplant or plant cell is constructed by incorporating one or more(several) expression constructs encoding a polypeptide into the planthost genome or chloroplast genome and propagating the resulting modifiedplant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide operably linked withappropriate regulatory sequences required for expression of thepolynucleotide in the plant or plant part of choice. Furthermore, theexpression construct may comprise a selectable marker useful foridentifying host cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide may be constitutive or inducible, or may be developmental,stage or tissue specific, and the gene product may be targeted to aspecific tissue or plant part such as seeds or leaves. Regulatorysequences are, for example, described by Tague et al., 1988, PlantPhysiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay inducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide in the plant. For instance, the promoterenhancer element may be an intron that is placed between the promoterand the polynucleotide encoding a polypeptide. For instance, Xu et al.,1993, supra, disclose the use of the first intron of the rice actin 1gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) and can alsobe used for transforming monocots, although other transformation methodsare often used for these plants. Presently, the method of choice forgenerating transgenic monocots is particle bombardment (microscopic goldor tungsten particles coated with the transforming DNA) of embryoniccalli or developing embryos (Christou, 1992, Plant J. 2: 275-281;Shimamoto, 1994, Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992,Bio/Technology 10: 667-674). An alternative method for transformation ofmonocots is based on protoplast transformation as described by Omirullehet al., 1993, Plant Mol. Biol. 21: 415-428. Additional transformationmethods for use in accordance with the present disclosure include thosedescribed in U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which areherein incorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct prepared according to the present invention, transgenicplants may be made by crossing a plant having the construct to a secondplant lacking the construct. For example, a construct encoding apolypeptide can be introduced into a particular plant variety bycrossing, without the need for ever directly transforming a plant ofthat given variety. Therefore, the present invention encompasses notonly a plant directly regenerated from cells which have been transformedin accordance with the present invention, but also the progeny of suchplants. As used herein, progeny may refer to the offspring of anygeneration of a parent plant prepared in accordance with the presentinvention. Such progeny may include a DNA construct prepared inaccordance with the present invention, or a portion of a DNA constructprepared in accordance with the present invention. Crossing results inthe introduction of a transgene into a plant line by cross pollinating astarting line with a donor plant line. Non-limiting examples of suchsteps are further articulated in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideof the present invention comprising: (a) cultivating a transgenic plantor a plant cell comprising a polynucleotide encoding the polypeptideunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Removal or Reduction of Cellobiohydrolase Activity

The present invention also relates to methods of producing a mutant of aparent cell, which comprises disrupting or deleting a polynucleotide, ora portion thereof, encoding a polypeptide of the present invention,which results in the mutant cell producing less of the polypeptide thanthe parent cell when cultivated under the same conditions.

The mutant cell may be constructed by reducing or eliminating expressionof the polynucleotide using methods well known in the art, for example,insertions, disruptions, replacements, or deletions. In a preferredaspect, the polynucleotide is inactivated. The polynucleotide to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required for theexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thepolynucleotide. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the polynucleotide may be performed bysubjecting the parent cell to mutagenesis and selecting for mutant cellsin which expression of the polynucleotide has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the polynucleotide may be accomplishedby introduction, substitution, or removal of one or more (several)nucleotides in the gene or a regulatory element required for thetranscription or translation thereof. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of the start codon, or a change in the open reading frame.Such modification or inactivation may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. Although, in principle, the modification may beperformed in vivo, i.e., directly on the cell expressing thepolynucleotide to be modified, it is preferred that the modification beperformed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of apolynucleotide is based on techniques of gene replacement, genedeletion, or gene disruption. For example, in the gene disruptionmethod, a nucleic acid sequence corresponding to the endogenouspolynucleotide is mutagenized in vitro to produce a defective nucleicacid sequence that is then transformed into the parent cell to produce adefective gene. By homologous recombination, the defective nucleic acidsequence replaces the endogenous polynucleotide. It may be desirablethat the defective polynucleotide also encodes a marker that may be usedfor selection of transformants in which the polynucleotide has beenmodified or destroyed. In a particularly preferred aspect, thepolynucleotide is disrupted with a selectable marker such as thosedescribed herein.

The present invention also relates to methods of inhibiting theexpression of a polypeptide having cellobiohydrolase activity in a cell,comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. In a preferredaspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length.

The dsRNA is preferably a small interfering RNA (sRNA) or a micro RNA(miRNA). In a preferred aspect, the dsRNA is small interfering RNA(siRNAs) for inhibiting transcription. In another preferred aspect, thedsRNA is micro RNA (miRNAs) for inhibiting translation.

The present invention also relates to such double-stranded RNA (dsRNA)molecules, comprising a portion of the mature polypeptide codingsequence of SEQ ID NO: 1 for inhibiting expression of the polypeptide ina cell. While the present invention is not limited by any particularmechanism of action, the dsRNA can enter a cell and cause thedegradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed to dsRNA,mRNA from the homologous gene is selectively degraded by a processcalled RNA interference (RNAi).

The dsRNAs of the present invention can be used in gene-silencing. Inone aspect, the invention provides methods to selectively degrade RNAusing a dsRNAi of the present invention. The process may be practiced invitro, ex vivo or in vivo. In one aspect, the dsRNA molecules can beused to generate a loss-of-function mutation in a cell, an organ or ananimal. Methods for making and using dsRNA molecules to selectivelydegrade RNA are well known in the art; see, for example, U.S. Pat. Nos.6,489,127; 6,506,559; 6,511,824; and 6,515,109.

The present invention further relates to a mutant cell of a parent cellthat comprises a disruption or deletion of a polynucleotide encoding thepolypeptide or a control sequence thereof or a silenced gene encodingthe polypeptide, which results in the mutant cell producing less of thepolypeptide or no polypeptide compared to the parent cell.

The polypeptide-deficient mutant cells are particularly useful as hostcells for the expression of native and heterologous polypeptides.Therefore, the present invention further relates to methods of producinga native or heterologous polypeptide, comprising: (a) cultivating themutant cell under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide. The term “heterologouspolypeptides” means polypeptides that are not native to the host cell,e.g., a variant of a native protein. The host cell may comprise morethan one copy of a polynucleotide encoding the native or heterologouspolypeptide.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallycellobiohydrolase-free product is of particular interest in theproduction of eukaryotic polypeptides, in particular fungal proteinssuch as enzymes. The cellobiohydrolase-deficient cells may also be usedto express heterologous proteins of pharmaceutical interest such ashormones, growth factors, receptors, and the like. The term “eukaryoticpolypeptides” includes not only native polypeptides, but also thosepolypeptides, e.g., enzymes, which have been modified by amino acidsubstitutions, deletions or additions, or other such modifications toenhance activity, thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from cellobiohydrolase activity that is produced by amethod of the present invention.

Compositions

The present invention also relates to compositions comprising apolypeptide having cellobiohydrolase activity of the present invention.Preferably, the compositions are enriched in such a polypeptide. Theterm “enriched” indicates that the cellulolytic enhancing activity ofthe composition has been increased, e.g., with an enrichment factor ofat least 1.1.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as one or more (several) enzymes selected from thegroup consisting of a cellulase, a hemicellulase, an expansin, anesterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

The present invention is also directed to the following methods for thepolypeptides of the present invention, or compositions thereof.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellobiohydrolase activity of the present invention. In oneaspect, the method above further comprises recovering the degraded orconverted cellulosic material. Soluble products of degradation orconversion of the cellulosic material can be separated from theinsoluble cellulosic material using technology well known in the artsuch as, for example, centrifugation, filtration, and gravity settling.

The present invention also relates to methods for producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellobiohydrolase activity of the present invention; (b)fermenting the saccharified cellulosic material with one or more(several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of apolypeptide having cellobiohydrolase activity of the present invention.In one aspect, the fermenting of the cellulosic material produces afermentation product. In another aspect, the method further comprisesrecovering the fermentation product from the fermentation.

The processing of the cellulosic material according to the presentinvention can be accomplished using processes conventional in the art.Moreover, the methods of the present invention can be implemented usingany conventional biomass processing apparatus configured to operate inaccordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and cofermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC). SHF uses separate process stepsto first enzymatically hydrolyze cellulosic material to fermentablesugars, e.g., glucose, cellobiose, cellotriose, and pentose sugars, andthen ferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of the cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the cofermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF involves a separate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more(several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd, L. R.,Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbialcellulose utilization: Fundamentals and biotechnology, Microbiol. Mol.Biol. Reviews 66: 506-577). It is understood herein that any methodknown in the art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof, can be usedin the practicing the methods of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza, Flavio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee,J. M., 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996,Enhancement of enzymatic cellulose hydrolysis using a novel type ofbioreactor with intensive stirring induced by electromagnetic field,Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor typesinclude: fluidized bed, upflow blanket, immobilized, and extruder typereactors for hydrolysis and/or fermentation.

Pretreatment.

In practicing the methods of the present invention, any pretreatmentprocess known in the art can be used to disrupt plant cell wallcomponents of the cellulosic material (Chandra et al., 2007, Substratepretreatment: The key to effective enzymatic hydrolysis oflignocellulosics? Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe andZacchi, 2007, Pretreatment of lignocellulosic materials for efficientbioethanol production, Adv. Biochem. Engin./Biotechnol. 108: 41-65;Hendriks and Zeeman, 2009, Pretreatments to enhance the digestibility oflignocellulosic biomass, Bioresource Technol. 100: 10-18; Mosier et al.,2005, Features of promising technologies for pretreatment oflignocellulosic biomass, Bioresource Technol. 96: 673-686; Taherzadehand Karimi, 2008, Pretreatment of lignocellulosic wastes to improveethanol and biogas production: A review, Int. J. of Mol. Sci. 9:1621-1651; Yang and Wyman, 2008, Pretreatment: the key to unlockinglow-cost cellulosic ethanol, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, pre-soaking, wetting, washing, and/or conditioning prior topretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, and gamma irradiationpretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of the cellulosicmaterial to fermentable sugars (even in absence of enzymes).

Steam Pretreatment: In steam pretreatment, cellulosic material is heatedto disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. Cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably done at 140-230° C., more preferably 160-200° C., and mostpreferably 170-190° C., where the optimal temperature range depends onany addition of a chemical catalyst. Residence time for the steampretreatment is preferably 1-15 minutes, more preferably 3-12 minutes,and most preferably 4-10 minutes, where the optimal residence timedepends on temperature range and any addition of a chemical catalyst.Steam pretreatment allows for relatively high solids loadings, so thatcellulosic material is generally only moist during the pretreatment. Thesteam pretreatment is often combined with an explosive discharge of thematerial after the pretreatment, which is known as steam explosion, thatis, rapid flashing to atmospheric pressure and turbulent flow of thematerial to increase the accessible surface area by fragmentation (Duffand Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi,2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent ApplicationNo. 20020164730). During steam pretreatment, hemicellulose acetyl groupsare cleaved and the resulting acid autocatalyzes partial hydrolysis ofthe hemicellulose to monosaccharides and oligosaccharides. Lignin isremoved to only a limited extent.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 3% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762).

Chemical Pretreatment The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Examples of suitable chemicalpretreatment processes include, for example, dilute acid pretreatment,lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX),ammonia percolation (APR), and organosolv pretreatments.

In dilute acid pretreatment, cellulosic material is mixed with diluteacid, typically H₂SO₄, and water to form a slurry, heated by steam tothe desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, supra; Schell et al., 2004, Bioresource Technol. 91:179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to, limepretreatment, wet oxidation, ammonia percolation (APR), and ammoniafiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium carbonate, sodium hydroxide,or ammonia at low temperatures of 85-150° C. and residence times from 1hour to several days (Wyman et al., 2005, Bioresource Technol. 96:1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO2006/110891, WO 2006/11899, WO 2006/11900, and WO 2006/110901 disclosepretreatment methods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed at preferably 1-40% drymatter, more preferably 2-30% dry matter, and most preferably 5-20% drymatter, and often the initial pH is increased by the addition of alkalisuch as sodium carbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion), can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-100°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). AFEX pretreatment results in the depolymerization ofcellulose and partial hydrolysis of hemicellulose. Lignin-carbohydratecomplexes are cleaved.

Organosolv pretreatment delignifies cellulosic material by extractionusing aqueous ethanol (40-60% ethanol) at 160-200° C. for 30-60 minutes(Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan et al., 2006,Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl. Biochem.Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose isremoved.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as anacid treatment, and more preferably as a continuous dilute and/or mildacid treatment. The acid is typically sulfuric acid, but other acids canalso be used, such as acetic acid, citric acid, nitric acid, phosphoricacid, tartaric acid, succinic acid, hydrogen chloride, or mixturesthereof. Mild acid treatment is conducted in the pH range of preferably1-5, more preferably 1-4, and most preferably 1-3. In one aspect, theacid concentration is in the range from preferably 0.01 to 20 wt % acid,more preferably 0.05 to 10 wt % acid, even more preferably 0.1 to 5 wt %acid, and most preferably 0.2 to 2.0 wt % acid. The acid is contactedwith cellulosic material and held at a temperature in the range ofpreferably 160-220° C., and more preferably 165-195° C., for periodsranging from seconds to minutes to, e.g., 1 second to 60 minutes.

In another aspect, pretreatment is carried out as an ammonia fiberexplosion step (AFEX pretreatment step).

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, cellulosic material is present during pretreatment inamounts preferably between 10-80 wt %, more preferably between 20-70 wt%, and most preferably between 30-60 wt %, such as around 50 wt %. Thepretreated cellulosic material can be unwashed or washed using anymethod known in the art, e.g., washed with water.

Mechanical Pretreatment: The term “mechanical pretreatment” refers tovarious types of grinding or milling (e.g., dry milling, wet milling, orvibratory ball milling).

Physical Pretreatment: The term “physical pretreatment” refers to anypretreatment that promotes the separation and/or release of cellulose,hemicellulose, and/or lignin from the cellulosic material. For example,physical pretreatment can involve irradiation (e.g., microwaveirradiation), steaming/steam explosion, hydrothermolysis, andcombinations thereof.

Physical pretreatment can involve high pressure and/or high temperature(steam explosion). In one aspect, high pressure means pressure in therange of preferably about 300 to about 600 psi, more preferably about350 to about 550 psi, and most preferably about 400 to about 500 psi,such as around 450 psi. In another aspect, high temperature meanstemperatures in the range of about 100 to about 300° C., preferablyabout 140 to about 235° C. In a preferred aspect, mechanicalpretreatment is performed in a batch-process, steam gun hydrolyzersystem that uses high pressure and high temperature as defined above,e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.

Combined Physical and Chemical Pretreatment: Cellulosic material can bepretreated both physically and chemically. For instance, thepretreatment step can involve dilute or mild acid treatment and hightemperature and/or pressure treatment. The physical and chemicalpretreatments can be carried out sequentially or simultaneously, asdesired. A mechanical pretreatment can also be included.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto mechanical, chemical, or physical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms (see, for example, Hsu, T.-A., 1996,Pretreatment of biomass, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212; Ghosh and Singh, 1993, Physicochemical and biologicaltreatments for enzymatic/microbial conversion of cellulosic biomass,Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreatinglignocellulosic biomass: a review, in Enzymatic Conversion of Biomassfor Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P.,eds., ACS Symposium Series 566, American Chemical Society, Washington,D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T.,1999, Ethanol production from renewable resources, in Advances inBiochemical Engineering/Biotechnology, Scheper, T., ed., Springer-VerlagBerlin Heidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996,Fermentation of lignocellulosic hydrolysates for ethanol production,Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990,Production of ethanol from lignocellulosic materials: State of the art,Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification.

In the hydrolysis step, also known as saccharification, the cellulosicmaterial, e.g., pretreated, is hydrolyzed to break down cellulose andalternatively also hemicellulose to fermentable sugars, such as glucose,cellobiose, xylose, xylulose, arabinose, mannose, galactose, and/orsoluble oligosaccharides. The hydrolysis is performed enzymatically byan enzyme composition in the presence of a polypeptide of the presentinvention. The enzyme and protein components of the compositions can beadded sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In a preferred aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the pretreated cellulosic material (substrate)is fed gradually to, for example, an enzyme containing hydrolysissolution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 96 hours, more preferably about 16 to about 72 hours,and most preferably about 24 to about 48 hours. The temperature is inthe range of preferably about 25° C. to about 70° C., more preferablyabout 30° C. to about 65° C., and more preferably about 40° C. to 60°C., in particular about 50° C. The pH is in the range of preferablyabout 3 to about 8, more preferably about 3.5 to about 7, and mostpreferably about 4 to about 6, in particular about pH 5. The dry solidscontent is in the range of preferably about 5 to about 50 wt %, morepreferably about 10 to about 40 wt %, and most preferably about 20 toabout 30 wt %.

The optimum amounts of the enzymes and polypeptides havingcellobiohydrolase activity depend on several factors including, but notlimited to, the mixture of component cellulolytic enzymes, thecellulosic substrate, the concentration of cellulosic substrate, thepretreatment(s) of the cellulosic substrate, temperature, time, pH, andinclusion of fermenting organism (e.g., yeast for SimultaneousSaccharification and Fermentation).

In one aspect, an effective amount of cellulolytic or hemicellulolyticenzyme protein to cellulosic material is about 0.5 to about 50 mg,preferably at about 0.5 to about 40 mg, more preferably at about 0.5 toabout 25 mg, more preferably at about 0.75 to about 20 mg, morepreferably at about 0.75 to about 15 mg, even more preferably at about0.5 to about 10 mg, and most preferably at about 2.5 to about 10 mg perg of cellulosic material.

In another aspect, an effective amount of polypeptide(s) havingcellobiohydrolase activity to cellulosic material is about 0.01 to about50.0 mg, preferably about 0.01 to about 40 mg, more preferably about0.01 to about 30 mg, more preferably about 0.01 to about 20 mg, morepreferably about 0.01 to about 10 mg, more preferably about 0.01 toabout 5 mg, more preferably at about 0.025 to about 1.5 mg, morepreferably at about 0.05 to about 1.25 mg, more preferably at about0.075 to about 1.25 mg, more preferably at about 0.1 to about 1.25 mg,even more preferably at about 0.15 to about 1.25 mg, and most preferablyat about 0.25 to about 1.0 mg per g of cellulosic material.

In another aspect, an effective amount of polypeptide(s) havingcellobiohydrolase activity to cellulolytic enzyme(s) is about 0.005 toabout 1.0 g, preferably at about 0.01 to about 1.0 g, more preferably atabout 0.15 to about 0.75 g, more preferably at about 0.15 to about 0.5g, more preferably at about 0.1 to about 0.5 g, even more preferably atabout 0.1 to about 0.5 g, and most preferably at about 0.05 to about 0.2g per g of cellulolytic enzyme(s).

In one aspect, the enzyme composition comprises or further comprises oneor more (several) proteins selected from the group consisting of acellulase, a GH61 polypeptide having cellulolytic enhancing activity, ahemicellulase, an expansin, an esterase, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin. Inanother aspect, the cellulase is preferably one or more (several)enzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase. In another aspect, thehemicellulase is preferably one or more (several) enzymes selected fromthe group consisting of an acetylmannan esterase, an acetyxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase.

In another aspect, the enzyme composition comprises one or more(several) cellulolytic enzymes. In another aspect, the enzymecomposition comprises or further comprises one or more (several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (several) cellulolytic enzymes and one or more(several) hemicellulolytic enzymes. In another aspect, the enzymecomposition comprises one or more (several) enzymes selected from thegroup of cellulolytic enzymes and hemicellulolytic enzymes. In anotheraspect, the enzyme composition comprises an endoglucanase. In anotheraspect, the enzyme composition comprises a cellobiohydrolase. In anotheraspect, the enzyme composition comprises a beta-glucosidase. In anotheraspect, the enzyme composition comprises a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises an endoglucanase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a cellobiohydrolase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a beta-glucosidase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises an endoglucanase and a cellobiohydrolase. Inanother aspect, the enzyme composition comprises an endoglucanase and abeta-glucosidase. In another aspect, the enzyme composition comprises acellobiohydrolase and a beta-glucosidase. In another aspect, the enzymecomposition comprises an endoglucanase, a cellobiohydrolase, and apolypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an endoglucanase, a beta-glucosidase,and a polypeptide having cellulolytic enhancing activity. In anotheraspect, the enzyme composition comprises a cellobiohydrolase, abeta-glucosidase, and a polypeptide having cellulolytic enhancingactivity. In another aspect, the enzyme composition comprises anendoglucanase, a cellobiohydrolase, and a beta-glucosidase, and apolypeptide having cellulolytic enhancing activity.

In another aspect, the enzyme composition comprises an acetylmannanesterase. In another aspect, the enzyme composition comprises anacetyxylan esterase. In another aspect, the enzyme composition comprisesan arabinanase (e.g., alpha-L-arabinanase). In another aspect, theenzyme composition comprises an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase). In another aspect, the enzyme compositioncomprises a coumaric acid esterase. In another aspect, the enzymecomposition comprises a feruloyl esterase. In another aspect, the enzymecomposition comprises a galactosidase (e.g., alpha-galactosidase and/orbeta-galactosidase). In another aspect, the enzyme composition comprisesa glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, theenzyme composition comprises a glucuronoyl esterase. In another aspect,the enzyme composition comprises a mannanase. In another aspect, theenzyme composition comprises a mannosidase (e.g., beta-mannosidase). Inanother aspect, the enzyme composition comprises a xylanase. In apreferred aspect, the xylanase is a Family 10 xylanase. In anotheraspect, the enzyme composition comprises a xylosidase (e.g.,beta-xylosidase). In another aspect, the enzyme composition comprises anexpansin. In another aspect, the enzyme composition comprises anesterase. In another aspect, the enzyme composition comprises a laccase.In another aspect, the enzyme composition comprises a ligninolyticenzyme. In a preferred aspect, the ligninolytic enzyme is a manganeseperoxidase. In another preferred aspect, the ligninolytic enzyme is alignin peroxidase. In another preferred aspect, the ligninolytic enzymeis a H₂O₂-producing enzyme. In another aspect, the enzyme compositioncomprises a pectinase. In another aspect, the enzyme compositioncomprises a peroxidase. In another aspect, the enzyme compositioncomprises a protease. In another aspect, the enzyme compositioncomprises a swollenin.

In the methods of the present invention, the enzyme(s) can be addedprior to or during fermentation, e.g., during saccharification or duringor after propagation of the fermenting microorganism(s).

One or more (several) components of the enzyme composition may bewild-type proteins, recombinant proteins, or a combination of wild-typeproteins and recombinant proteins. For example, one or more (several)components may be native proteins of a cell, which is used as a hostcell to express recombinantly one or more (several) other components ofthe enzyme composition. One or more (several) components of the enzymecomposition may be produced as monocomponents, which are then combinedto form the enzyme composition. The enzyme composition may be acombination of multicomponent and monocomponent protein preparations.

The enzymes used in the methods of the present invention may be in anyform suitable for use, such as, for example, a crude fermentation brothwith or without cells removed, a cell lysate with or without cellulardebris, a semi-purified or purified enzyme preparation, or a host cellas a source of the enzymes. The enzyme composition may be a dry powderor granulate, a non-dusting granulate, a liquid, a stabilized liquid, ora stabilized protected enzyme. Liquid enzyme preparations may, forinstance, be stabilized by adding stabilizers such as a sugar, a sugaralcohol or another polyol, and/or lactic acid or another organic acidaccording to established processes.

The enzymes can be derived or obtained from any suitable origin,including, bacterial, fungal, yeast, plant, or mammalian origin. Theterm “obtained” means herein that the enzyme may have been isolated froman organism that naturally produces the enzyme as a native enzyme. Theterm “obtained” also means herein that the enzyme may have been producedrecombinantly in a host organism employing methods described herein,wherein the recombinantly produced enzyme is either native or foreign tothe host organism or has a modified amino acid sequence, e.g., havingone or more (several) amino acids that are deleted, inserted and/orsubstituted, i.e., a recombinantly produced enzyme that is a mutantand/or a fragment of a native amino acid sequence or an enzyme producedby nucleic acid shuffling processes known in the art. Encompassed withinthe meaning of a native enzyme are natural variants and within themeaning of a foreign enzyme are variants obtained recombinantly, such asby site-directed mutagenesis or shuffling.

The polypeptide having enzyme activity may be a bacterial polypeptide.For example, the polypeptide may be a gram positive bacterialpolypeptide such as a Bacillus, Streptococcus, Streptomyces,Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium,Geobacillus, or Oceanobacillus polypeptide having enzyme activity, or aGram negative bacterial polypeptide such as an E. coli, Pseudomonas,Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium,Ilyobacter, Neisseria, or Ureaplasma polypeptide having enzyme activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having enzyme activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having enzymeactivity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide having enzymeactivity.

The polypeptide having enzyme activity may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having enzyme activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having enzymeactivity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasfi, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having enzymeactivity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminurn, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulaturn, Fusarium roseum, Fusarium sambucinum, Fusariumsarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusariumtorulosum, Fusarium trichothecioides, Fusarium venenatum, Humicolagrisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucormiehei, Myceliophthora thermophila, Neurospora crassa, Penicilliumfuniculosum, Penicillium purpurogenum, Phanerochaete chtysosporium,Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa,Thielavia australeinsis, Thielavia fimeti, Thielavia microspora,Thielavia ovispora, Thielavia peruviana, Thielavia spededonium,Thielavia setosa, Thielavia subthermophila, Thielavia terrestris,Trichoderma harzianurn, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaeasaccata polypeptide having enzyme activity.

Chemically modified or protein engineered mutants of the polypeptideshaving enzyme activity may also be used.

One or more (several) components of the enzyme composition may be arecombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host is preferably a heterologous host (enzyme isforeign to host), but the host may under certain conditions also be ahomologous host (enzyme is native to host). Monocomponent cellulolyticenzymes may also be prepared by purifying such a protein from afermentation broth.

In one aspect, the one or more (several) cellulolytic enzymes comprise acommercial cellulolytic enzyme preparation. Examples of commercialcellulolytic enzyme preparations suitable for use in the presentinvention include, for example, CELLIC™ CTec (Novozymes A/S), CELLIC™CTec2 (Novozymes A/S), CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188(Novozymes A/S), CELLUZYME™ (Novozymes A/S), CEREFLO™ (Novozymes A/S),and ULTRAFLO™ (Novozymes A/S), ACCELERASE™ (Genencor Int.), LAMINEX™(Genencor Int.), SPEZYME™ CP (Genencor Int.), ROHAMENT™ 7069 W (RöhmGmbH), FIBREZYME® LDI (Dyadic International, Inc.), FIBREZYME® LBR(Dyadic International, Inc.), or VISCOSTAR® 150 L (Dyadic International,Inc.). The cellulase enzymes are added in amounts effective from about0.001 to about 5.0 wt % of solids, more preferably from about 0.025 toabout 4.0 wt % of solids, and most preferably from about 0.005 to about2.0 wt % of solids.

Examples of bacterial endoglucanases that can be used in the methods ofthe present invention, include, but are not limited to, an Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263; Trichodermareesei Cel7B endoglucanase I; GENBANK™ accession no. M15665);Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene63:11-22; Trichoderma reesei Cel5A endoglucanase II; GENBANK™ accessionno. M19373); Trichoderma reesei endoglucanase III (Okada et al., 1988,Appl. Environ. Microbiol. 64: 555-563; GENBANK™ accession no. AB003694);Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, MolecularMicrobiology 13: 219-228; GENBANK™ accession no. Z33381); Aspergillusaculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18:5884); Aspergillus kawachii endoglucanase (Sakamoto et al., 1995,Current Genetics 27: 435-439); Erwinia carotovara endoglucanase(Saarilahti et al., 1990, Gene 90: 9-14); Fusarium oxysporumendoglucanase (GENBANK™ accession no. L29381); Humicola grisea var.thermoidea endoglucanase (GENBANK™ accession no. AB003107); Melanocarpusalbomyces endoglucanase (GENBANK™ accession no. MAL515703); Neurosporacrassa endoglucanase (GENBANK™ accession no. XM_(—)324477); Humicolainsolens endoglucanase V); Myceliophthora thermophila CBS117.65endoglucanase; basidiomycete CBS 495.95 endoglucanase; basidiomycete CBS494.95 endoglucanase; Thielavia terrestris NRRL 8126 CEL6Bendoglucanase; Thielavia terrestris NRRL 8126 CEL6C endoglucanase;Thielavia terrestris NRRL 8126 CEL7C endoglucanase; Thielavia terrestrisNRRL 8126 CEL7E endoglucanase; Thielavia terrestris NRRL 8126 CEL7Fendoglucanase; Cladorrhinum foecundissimum ATCC 62373 CEL7Aendoglucanase; and Trichoderma reesei strain No. VTT-D-80133endoglucanase (GENBANK™ accession no. M15665). Examples ofcellobiohydrolases useful in the present invention include, but are notlimited to, Trichoderma reesei cellobiohydrolase I; Trichoderma reeseicellobiohydrolase II; Humicola insolens cellobiohydrolase I);Myceliophthora thermophila cellobiohydrolase II; Thielavia terrestriscellobiohydrolase II (CEL6A); Chaetomium thermophilum cellobiohydrolaseI; and Chaetomium thermophilum cellobiohydrolase II, Aspergillusfumigatus cellobiohydrolase I, and Aspergillus fumigatuscellobiohydrolase II.

Examples of beta-glucosidases useful in the present invention include,but are not limited to, Aspergillus oryzae beta-glucosidase; Aspergillusfumigatus beta-glucosidase; Penicillium brasilianum IBT 20888beta-glucosidase; Aspergillus niger beta-glucosidase; and Aspergillusaculeatus beta-glucosidase. The Aspergillus oryzae beta-glucosidase canbe obtained according to WO 2002/095014. The Aspergillus fumigatusbeta-glucosidase can be obtained according to WO 2005/047499. ThePenicillium brasilianum beta-glucosidase can be obtained according to WO2007/019442. The Aspergillus niger beta-glucosidase can be obtainedaccording to Dan et al., 2000, J. Biol. Chem. 275: 4973-4980. TheAspergillus aculeatus beta-glucosidase can be obtained according toKawaguchi et al., 1996, Gene 173: 287-288.

The beta-glucosidase may be a fusion protein. In one aspect, thebeta-glucosidase is the Aspergillus oryzae beta-glucosidase variant BGfusion protein or the Aspergillus oryzae beta-glucosidase fusion proteinobtained according to WO 2008/057637.

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Other cellulolytic enzymes that may be useful in the present inventionare described in EP 495,257, EP 531,315, EP 531,372, WO 89/09259, WO94/07998, WO 95/24471, WO 96/11262, WO 96/29397, WO 96/034108, WO97/14804, WO 98/08940, WO 98/012307, WO 98/13465, WO 98/015619, WO98/015633, WO 98/028411, WO 99/06574, WO 99/10481, WO 99/025846, WO99/025847, WO 99/031255, WO 2000/009707, WO 2002/050245, WO2002/0076792, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO2006/117432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO2008/008793, U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,457,046, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,691,178, U.S.Pat. No. 5,763,254, and U.S. Pat. No. 5,776,757.

In the methods of the present invention, any polypeptide havingcellulolytic enhancing activity can be used.

In a first aspect, the polypeptide having cellulolytic enhancingactivity comprises the following motifs:

[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ](SEQ ID NO: 27 or SEQ ID NO: 28) and [FW]-[TF]-K-[AIV],

wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5contiguous positions, and X(4) is any amino acid at 4 contiguouspositions.

The polypeptide comprising the above-noted motifs may further comprise:

H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 29 or SEQ ID NL: 30),[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 31), orH-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 29 or SEQ ID NO: 30) and[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 31),

wherein X is any amino acid, X(1,2) is any amino acid at 1 position or 2contiguous positions, X(3) is any amino acid at 3 contiguous positions,and X(2) is any amino acid at 2 contiguous positions. In the abovemotifs, the accepted IUPAC single letter amino acid abbreviation isemployed.

In a preferred aspect, the polypeptide having cellulolytic enhancingactivity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 29or SEQ ID NO: 30). In another preferred aspect, the isolated polypeptidehaving cellulolytic enhancing activity further comprises[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 31). In anotherpreferred aspect, the polypeptide having cellulolytic enhancing activityfurther comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 29 or SEQID NO: 30) and [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 31).

In a second aspect, the polypeptide having cellulolytic enhancingactivity comprises the following motif:

[ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)-A- [HNQ](SEQ ID NO: 32 or SEQ ID NO: 33),

wherein x is any amino acid, x(4,5) is any amino acid at 4 or 5contiguous positions, and x(3) is any amino acid at 3 contiguouspositions. In the above motif, the accepted IUPAC single letter aminoacid abbreviation is employed.

Examples of polypeptides having cellulolytic enhancing activity usefulin the methods of the present invention include, but are not limited to,polypeptides having cellulolytic enhancing activity from Thielaviaterrestris (WO 2005/074647); polypeptides having cellulolytic enhancingactivity from Thermoascus aurantiacus (WO 2005/074656); polypeptideshaving cellulolytic enhancing activity from Trichoderma reesei (WO2007/089290); and polypeptides having cellulolytic enhancing activityfrom Myceliophthora thermophila (WO 2009/085935, WO 2009/085859, WO2009/085864, and WO 2009/085868). WO 2008/151043 discloses methods ofincreasing the activity of a polypeptide having cellulolytic enhancingactivity by adding a soluble activating divalent metal cation to acomposition comprising the polypeptide having cellulolytic enhancingactivity.

In one aspect, the one or more (several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes A/S),CELLIC™ HTec (Novozymes A/S), CELLIC™ HTec2 (Novozymes A/S), VISCOZYME®(Novozymes A/S), ULTRAFLO® (Novozymes A/S), PULPZYME® HC (NovozymesA/S), MULTIFECT® Xylanase (Genencor), ECOPULP® TX-200A (AB Enzymes), HSP6000 Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit, Wales, UK), DEPOL™740L. (Biocatalysts Limit, Wales, UK), and DEPOL™ 762P (BiocatalystsLimit, Wales, UK).

Examples of xylanases useful in the methods of the present inventioninclude, but are not limited to, Aspergillus aculeatus xylanase(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus xylanases (WO2006/078256), and Thielavia terrestris NRRL 8126 xylanases (WO2009/079210).

Examples of beta-xylosidases useful in the methods of the presentinvention include, but are not limited to, Trichoderma reeseibeta-xylosidase (UniProtKB/TrEMBL accession number Q92458), Talaromycesemersonii (SwissProt accession number Q8×212), and Neurospora crassa(SwissProt accession number Q7SOW4).

Examples of acetylxylan esterases useful in the methods of the presentinvention include, but are not limited to, Hypocrea jecorina acetylxylanesterase (WO 2005/001036), Neurospora crassa acetylxylan esterase(UniProt accession number q7s259), Thielavia terrestris NRRL 8126acetylxylan esterase (WO 2009/042846), Chaetomium globosum acetylxylanesterase (Uniprot accession number Q2GWX4), Chaetomium gracileacetylxylan esterase (GeneSeqP accession number AAB82124), Phaeosphaerianodorum acetylxylan esterase (Uniprot accession number Q0UHJ1), andHumicola insolens DSM 1800 acetylxylan esterase (WO 2009/073709).

Examples of ferulic acid esterases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800feruloyl esterase (WO 2009/076122), Neurospora crassa feruloyl esterase(UniProt accession number Q9HGR3), and Neosartorya fischeri feruloylesterase (UniProt Accession number A1D9T4).

Examples of arabinofuranosidases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800arabinofuranosidase (WO 2009/073383) and Aspergillus nigerarabinofuranosidase (GeneSeqP accession number AAR94170).

Examples of alpha-glucuronidases useful in the methods of the presentinvention include, but are not limited to, Aspergillus clavatusalpha-glucuronidase (UniProt accession number alcc12), Trichodermareesei alpha-glucuronidase (Uniprot accession number Q99024),Talaromyces emersonii alpha-glucuronidase (UniProt accession numberQ8×211), Aspergillus niger alpha-glucuronidase (Uniprot accession numberQ96WX9), Aspergillus terreus alpha-glucuronidase (SwissProt accessionnumber Q0CJP9), and Aspergillus fumigatus alpha-glucuronidase (SwissProtaccession number Q4WW45).

The enzymes and proteins used in the methods of the present inventionmay be produced by fermentation of the above-noted microbial strains ona nutrient medium containing suitable carbon and nitrogen sources andinorganic salts, using procedures known in the art (see, e.g., Bennett,J. W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, AcademicPress, CA, 1991). Suitable media are available from commercial suppliersor may be prepared according to published compositions (e.g., incatalogues of the American Type Culture Collection). Temperature rangesand other conditions suitable for growth and enzyme production are knownin the art (see, e.g., Bailey, J. E., and Ollis, D. F., BiochemicalEngineering Fundamentals, McGraw-Hill Book Company, NY, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme. Fermentation may, therefore,be understood as comprising shake flask cultivation, or small- orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the enzymeto be expressed or isolated. The resulting enzymes produced by themethods described above may be recovered from the fermentation mediumand purified by conventional procedures.

Fermentation.

The fermentable sugars obtained from the hydrolyzed cellulosic materialcan be fermented by one or more (several) fermenting microorganismscapable of fermenting the sugars directly or indirectly into a desiredfermentation product. “Fermentation” or “fermentation process” refers toany fermentation process or any process comprising a fermentation step.Fermentation processes also include fermentation processes used in theconsumable alcohol industry (e.g., beer and wine), dairy industry (e.g.,fermented dairy products), leather industry, and tobacco industry. Thefermentation conditions depend on the desired fermentation product andfermenting organism and can easily be determined by one skilled in theart.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous, as described herein.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be C₆ and/or C₅ fermenting organisms, or a combinationthereof. Both C₆ and C₅ fermenting organisms are well known in the art.Suitable fermenting microorganisms are able to ferment, i.e., convert,sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose,galactose, or oligosaccharides, directly or indirectly into the desiredfermentation product.

Examples of bacterial and fungal fermenting organisms producing ethanolare described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69:627-642.

Examples of fermenting microorganisms that can ferment C₆ sugars includebacterial and fungal organisms, such as yeast. Preferred yeast includesstrains of the Saccharomyces spp., preferably Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment C₅ sugars includebacterial and fungal organisms, such as some yeast. Preferred C₅fermenting yeast include strains of Pichia, preferably Pichia stipitis,such as Pichia stipitis CBS 5773; strains of Candida, preferably Candidaboidinii, Candida brassicae, Candida sheatae, Candida diddensii, Candidapseudotropicalis, or Candida utilis.

Other fermenting organisms include strains of Zymomonas, such asZymomonas mobilis; Hansenula, such as Hansenula anomala; Kluyveromyces,such as K. fragilis; Schizosaccharomyces, such as S. pombe; E. coli,especially E. coli strains that have been genetically modified toimprove the yield of ethanol; Clostridium, such as Clostridiumacetobutylicum, Chlostridium thermocellum, and Chlostridiumphytofermentans; Geobacillus sp.; Thermoanaerobacter, such asThermoanaerobacter saccharolyticum; and Bacillus, such as Bacilluscoagulans.

In a preferred aspect, the yeast is a Saccharomyces spp. In a morepreferred aspect, the yeast is Saccharomyces cerevisiae. In another morepreferred aspect, the yeast is Saccharomyces distaticus. In another morepreferred aspect, the yeast is Saccharomyces uvarum. In anotherpreferred aspect, the yeast is a Kluyveromyces. In another morepreferred aspect, the yeast is Kluyveromyces marxianus. In another morepreferred aspect, the yeast is Kluyveromyces fragilis. In anotherpreferred aspect, the yeast is a Candida. In another more preferredaspect, the yeast is Candida boidinii. In another more preferred aspect,the yeast is Candida brassicae. In another more preferred aspect, theyeast is Candida diddensii. In another more preferred aspect, the yeastis Candida pseudotropicalis. In another more preferred aspect, the yeastis Candida utilis. In another preferred aspect, the yeast is aClavispora. In another more preferred aspect, the yeast is Clavisporalusitaniae. In another more preferred aspect, the yeast is Clavisporaopuntiae. In another preferred aspect, the yeast is a Pachysolen. Inanother more preferred aspect, the yeast is Pachysolen tannophilus. Inanother preferred aspect, the yeast is a Pichia. In another morepreferred aspect, the yeast is a Pichia stipitis. In another preferredaspect, the yeast is a Bretannomyces. In another more preferred aspect,the yeast is Bretannomyces clausenii (Philippidis, G. P., 1996,Cellulose bioconversion technology, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212).

Bacteria that can efficiently ferment hexose and pentose to ethanolinclude, for example, Zymomonas mobilis, Clostridium acetobutylicum,Clostridium thermocellum, Chlostridium phytofermentans, Geobacillus sp.,Thermoanaerobacter saccharolyticum, and Bacillus coagulans (Philippidis,1996, supra).

In a preferred aspect, the bacterium is a Zymomonas. In a more preferredaspect, the bacterium is Zymomonas mobilis. In another preferred aspect,the bacterium is a Clostridium. In another more preferred aspect, thebacterium is Clostridium thermocellum.

Commercially available yeast suitable for ethanol production includes,e.g., ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™ (Fleischmann'sYeast, USA), SUPERSTART™ and THERMOSACC™ fresh yeast (EthanolTechnology, WI, USA), BIOFERM™ AFT and XR(NABC—North AmericanBioproducts Corporation, GA, USA), GERT STRAND™ (Gert Strand AB,Sweden), and FERMIOL™ (DSM Specialties).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (cofermentation) (Chen and Ho, 1993, Cloning andimproving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TALI genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Saccharomyces cerevisiae. In another preferred aspect, thegenetically modified fermenting microorganism is Zymomonas mobilis. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Escherichia coli. In another preferred aspect, thegenetically modified fermenting microorganism is Klebsiella oxytoca. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Kluyveromyces sp.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedlignocellulose or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, such as about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., inparticular about 32° C. or 50° C., and at about pH 3 to about pH 8, suchas around pH 4-5, 6, or 7.

In a preferred aspect, the yeast and/or another microorganism is appliedto the degraded cellulosic material and the fermentation is performedfor about 12 to about 96 hours, such as typically 24-60 hours. In apreferred aspect, the temperature is preferably between about 20° C. toabout 60° C., more preferably about 25° C. to about 50° C., and mostpreferably about 32° C. to about 50° C., in particular about 32° C. or50° C., and the pH is generally from about pH 3 to about pH 7,preferably around pH 4-7. However, some fermenting organisms, e.g.,bacteria, have higher fermentation temperature optima. Yeast or anothermicroorganism is preferably applied in amounts of approximately 10⁵ to10¹², preferably from approximately 10⁷ to 10¹⁰, especiallyapproximately 2×10⁸ viable cell count per ml of fermentation broth.Further guidance in respect of using yeast for fermentation can be foundin, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which ishereby incorporated by reference.

For ethanol production, following the fermentation the fermented slurryis distilled to extract the ethanol. The ethanol obtained according tothe methods of the invention can be used as, e.g., fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products:

A fermentation product can be any substance derived from thefermentation. The fermentation product can be, without limitation, analcohol (e.g., arabinitol, butanol, ethanol, glycerol, methanol,1,3-propanediol, sorbitol, and xylitol); an organic acid (e.g., aceticacid, acetonic acid, adipic acid, ascorbic acid, citric acid,2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid,gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid,itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid,oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); aketone (e.g., acetone); an amino acid (e.g., aspartic acid, glutamicacid, glycine, lysine, serine, and threonine); and a gas (e.g., methane,hydrogen (H₂), carbon dioxide (CO₂), and carbon monoxide (CO)). Thefermentation product can also be protein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is arabinitol. In another more preferred aspect, the alcohol isbutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is glycerol. In another morepreferred aspect, the alcohol is methanol. In another more preferredaspect, the alcohol is 1,3-propanediol. In another more preferredaspect, the alcohol is sorbitol. In another more preferred aspect, thealcohol is xylitol. See, for example, Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira, M.M., and Jonas, R., 2002, The biotechnological production of sorbitol,Appl. Microbiol. Biotechnol. 59: 400-408; Nigam, P., and Singh, D.,1995, Processes for fermentative production of xylitol—a sugarsubstitute, Process Biochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi,N. and Blaschek, H. P., 2003, Production of acetone, butanol and ethanolby Clostridium beijerinckii BA101 and in situ recovery by gas stripping,World Journal of Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard, A., andMargaritis, A., 2004, Empirical modeling of batch fermentation kineticsfor poly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

Recovery.

The fermentation product(s) can be optionally recovered from thefermentation medium using any method known in the art including, but notlimited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

Signal Peptide

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to16 of SEQ ID NO: 2. The polynucleotide may further comprise a geneencoding a protein, which is operably linked to the signal peptide. Theprotein is preferably foreign to the signal peptide. In one aspect, thepolynucleotide for the signal peptide is nucleotides 61 to 108 of SEQ IDNO: 1.

The present invention also relates to nucleic acid constructs,expression vectors and recombinant host cells comprising such apolynucleotide.

The present invention also relates to methods of producing a protein,comprising: (a) cultivating a recombinant host cell comprising such apolynucleotide; and (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andpolypeptides. The term “protein” also encompasses two or morepolypeptides combined to form the encoded product. The proteins alsoinclude hybrid polypeptides and fused polypeptides.

Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.For example, the protein may be an oxidoreductase, transferase,hydrolase, lyase, isomerase, or ligase such as an aminopeptidase,amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, another lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Media

MEX-1 medium was composed of 20 g of soya bean meal, 15 g of wheat bran,10 g of microcrystalline cellulose (AVICEL®; FMC, Philadelphia, Pa.,USA), 5 g of maltodextrin, 3 g of Bactopeptone, 0.2 g of pluronic, 1 gof olive oil, and deionized water to 1 liter.

PDA plates were composed of 39 grams of potato dextrose agar anddeionized water to 1 liter.

LB medium was composed of 10 g of tryptone, 5 g of yeast extract, 5 g ofsodium chloride, and deionized water to 1 liter.

LB plates were composed of 15 g of Bacto agar per liter of LB medium.

SOC medium was composed of 2% tryptone, 0.5% yeast extract, 10 mM NaCl,2.5 mM KCl, 10 mM MgCl₂, and 10 mM MgSO₄, sterilized by autoclaving andthen filter-sterilized glucose was added to 20 mM.

COVE N medium was composed of 218 g of sorbitol, 50 ml of COVE saltsolution, 10 g of dextrose, 2.02 g of KNO₃, 25 g of agar, and deionizedwater to 1 liter.

COVE salt solution was composed of 26 g of MgSO₄.7H₂O, 26 g of KCl, 76 gof KH₂PO₄, 50 ml of COVE trace metals solution, and deionized water to 1liter.

COVE trace metals solution was composed of 0.04 g Na₂B₄O₇.10H₂O, 0.4 gof CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g of MnSO₄.H₂O, 0.8 g ofNa₂MoO₄.2H₂O, 10 g of ZnSO₄.7H₂O, and deionized water to 1 liter.

SY50 medium was composed per liter of 50 g of sucrose, 2 g ofMgSO₄.7H2O, 10 g of KH₂PO₄, anhydrous, 2 g of K₂SO₄, 2 g of citric acid,10 g of yeast extract, 2 g of urea, 0.5 g of CaCl₂.2H₂O, and 0.5 g of200×AMG trace metals solution, pH 6.0.

200×AMG trace metals solution was composed per liter of 3 g of citricacid, 14.3 g of ZnSO₄.7H₂O, 2.5 g of CuSO₄.5H₂O, 13.8 g of FeSO₄.7H₂O,and 8.5 g of MnSO₄.H₂O.

Example 1 Preparation of Trichophaea saccata Strain CBS 804.70 Myceliafor cDNA Library Production

Trichophaea saccata CBS 804.70 was inoculated onto a PDA plate andincubated for 7 days at 28° C. Several mycelia-PDA agar plugs wereinoculated into 750 ml shake flasks containing 100 ml of MEX-1 medium.The flasks were incubated at 37° C. for 9 days with shaking at 150 rpm.The fungal mycelia were harvested by filtration through MIRACLOTH®(Calbiochem, San Diego, Calif., USA) before being frozen in liquidnitrogen. The mycelia were then pulverized into a powder by milling thefrozen mycelia together with an equal volume of dry ice in a coffeegrinder precooled with liquid nitrogen. The powder was transferred intoa liquid nitrogen prechilled mortar and pestle and ground to a finepowder with a small amount of baked quartz sand. The powdered mycelialmaterial was kept at −80° C. until use.

Example 2 Trichophaea saccata Strain CBS 804.70 RNA Isolation

Total RNA was prepared from the frozen, powdered mycelia of Trichophaeasaccata CBS 804.70 by extraction with guanidium thiocyanate followed byultracentrifugation through a 5.7 M CsCl cushion according to Chirgwinet al., 1979, Biochemistry 18: 5294-5299. The polyA enriched RNA wasisolated by oligo (dT)-cellulose affinity chromatography according toAviv et al., 1972, Proc. Natl. Acad. Sci. USA 69: 1408-1412.

Example 3 Construction of a Trichophaea saccata Strain CBS 804.70 cDNALibrary

Double stranded cDNA was synthesized according to the general methods ofGubler and Hoffman, 1983, Gene 25: 263-269; Sambrook, J., Fritsch, E.F., and Maniantis, T. Molecular cloning: A Laboratory Manual, 2^(nd)ed., 1989, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; andKofod et al., 1994, J. Biol. Chem. 269: 29182-29189, using a polyA-Not Iprimer (Promega Corp., Madison, Wis., USA). After synthesis, the cDNAwas treated with mung bean nuclease, blunt ended with T4 DNA polymerase,and ligated to a 50-fold molar excess of Eco RI adaptors (InvitrogenCorp., Carlsbad, Calif., USA). The cDNA was cleaved with Not I and thecDNA was size fractionated by 0.8% agarose gel electrophoresis using in44 mM Tris base, 44 mM boric acid, 0.5 mM EDTA (TBE) buffer. Thefraction of cDNA of 700 bp and larger was excised from the gel andpurified using a GFX® PCR DNA and Gel Band Purification Kit (GEHealthcare, United Kingdom) according to the manufacturer'sinstructions.

The prepared cDNA was then directionally cloned by ligation into EcoRI-Not I cleaved pMHas5 (WO 03/044049) using a Rapid Ligation Kit (RocheDiagnostics GmbH, Penzberg, Germany) according to the manufacturer'sinstructions. The ligation mixture was electroporated into E. coli DH10Bcells (Invitrogen Corp., Carlsbad, Calif., USA) using a GENE PULSER® andPulse Controller (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) at50 μF, 25 mAmp, 1.8 kV with a 2 mm gap cuvette according to themanufacturer's procedure.

The electroporated cells were spread onto LB plates supplemented with 50μg of kanamycin per ml. A cDNA plasmid pool was prepared fromapproximately 30,000 total transformants of the original cDNA-pMHas5vector ligation. Plasmid DNA was prepared directly from the pool ofcolonies using a QIAPREP® Spin Midi/Maxiprep Kit (QIAGEN GmbHCorporation, Hilden, Germany). The cDNA library was designated SBL521-2.

Example 4 Construction of a SigA4 Transposon Containing the β-LactamaseReporter Gene

A transposon containing plasmid designated pSigA4 was constructed fromthe pSigA2 transposon containing plasmid described in WO 01/77315 inorder to create an improved version of the signal trapping transposon ofpSigA2 with decreased selection background. The pSigA2 transposoncontains a signal-less beta-lactamase construct encoded on thetransposon itself. PCR was used to create a deletion of the intactbeta-lactamase gene found on the plasmid backbone using a proofreadingPROOFSTART® DNA polymerase (QIAGEN GmbH Corporation, Hilden, Germany)and the following 5′ phosphorylated primers (TAG Copenhagen, Denmark):

SigA2NotU-P: (SEQ ID NO: 3) 5′-TCGCGATCCGTTTTCGCATTTATCGTGAAACGCT-3′SigA2NotD-P: (SEQ ID NO: 4) 5′-CCGCAAACGCTGGTGAAAGTAAAAGATGCTGAA-3′

The amplification reaction was composed of 1 μl of pSigA2 (10 ng/μl), 5μl of 10× PROOFSTART® Buffer (QIAGEN GmbH Corporation, Hilden, Germany),2.5 μl of dNTP mix (20 mM), 0.5 μl of SigA2NotU-P (10 mM), 0.5 μl ofSigA2NotD-P (10 mM), 10 μl of Q solution (QIAGEN GmbH Corporation,Hilden, Germany), and 31.25 μl of deionized water. A DNA ENGINE™ ThermalCycler (MJ Research Inc., Waltham, Mass., USA) was used for theamplification programmed for 1 cycle at 95° C. for 5 minutes; and 20cycles each at 94° C. for 30 seconds, 62° C. for 30 seconds, and 72° C.for 4 minutes.

A 3.9 kb PCR reaction product was isolated by 0.8% agarose gelelectrophoresis using 40 mM Tris base-20 mM sodium acetate-1 mM disodiumEDTA (TAE) buffer and 0.1 μg of ethidium bromide per ml. The DNA bandwas visualized with the aid of an Eagle Eye Imaging System (Stratagene,La Jolla, Calif., USA) at 360 nm. The 3.9 kb DNA band was excised fromthe gel and purified by using a GFX® PCR DNA and Gel Band PurificationKit according to the manufacturer's instructions.

The 3.9 kb fragment was self-ligated at 16° C. overnight with 10 unitsof T4 DNA ligase (New England Biolabs, Inc., Beverly, Mass., USA), 9 μlof the 3.9 kb PCR fragment, and 1 μl of 10× ligation buffer (New EnglandBiolabs, Inc., Beverly, Mass., USA). The ligation was heat inactivatedfor 10 minutes at 65° C. and then digested with Dpn I at 37° C. for 2hours. After incubation, the digestion was purified using a GFX® PCR DNAand Gel Band Purification Kit.

The purified material was then transformed into E. coli Top10 competentcells (Invitrogen Corp., Carlsbad, Calif., USA) according to themanufacturer's instructions. The transformation mixture was plated ontoLB plates supplemented with 25 μg of chloramphenicol per ml. Plasmidminipreps were prepared from several transformants and digested with BglII. One plasmid with the correct construction was chosen. The plasmidwas designated pSigA4. Plasmid pSigA4 contains the Bgl II flankedtransposon SigA2 identical to that disclosed in WO 01/77315.

A 60 μl sample of plasmid pSigA4 DNA (0.3 μg/μl) was digested with BglII and separated by 0.8% agarose gel electrophoresis using TAE buffer. ASigA2 transposon DNA band of 2 kb was eluted with 200 μl of EB buffer(QIAGEN GmbH Corporation, Hilden, Germany) and purified using a GFX® PCRDNA and Gel Band Purification Kit according to the manufacturer'sinstructions and eluted in 200 μl of EB buffer. SigA2 was used fortransposon assisted signal trapping.

Example 5 Transposon Assisted Signal Trapping of Trichophaea saccata CBS804.70

A complete description of transposon assisted signal trapping can befound in WO 01/77315. A cDNA plasmid pool was prepared from 30,000 totaltransformants of the original cDNA-pMHas5 vector ligation. Plasmid DNAwas prepared directly from a pool of colonies recovered from solid LBselective medium using a QIAPREP® Spin Midi/Maxiprep Kit. The plasmidpool was treated with transposon SigA2 and MuA transposase (Finnzymes OY, Espoo, Finland) according to the manufacturer's instructions.

For in vitro transposon tagging of the Trichophaea saccata CBS 804.70cDNA library, 4 or 8 μl of SigA2 transposon containing approximately 2.6μg of DNA were mixed with 1 μl of the plasmid DNA pool of theTrichophaea saccata CBS 804.70 cDNA library containing 2 μg of DNA, 2 μlof MuA transposase (0.22 μg/μl), and 5 μl of 5× buffer (Finnzymes O Y,Espoo, Finland) in a total volume of 50 μl and incubated at 30° C. for3.5 hours followed by heat inactivation at 75° C. for 10 minutes. TheDNA was precipitated by addition of 5 μl of 3 M sodium acetate pH 5 and110 μl of 96% ethanol and centrifuged for 30 minutes at 10,000× g. Thepellet was washed in 70% ethanol, air dried at room temperature, andresuspended in 10 μl of 10 mM Tris, pH 8, 1 mM EDTA (TE) buffer.

A 1.5 μl volume of the transposon tagged plasmid pool was electroporatedinto 20 μl of E. coli DH10B ultracompetent cells (Gibco-BRL,Gaithersburg Md., USA) according to the manufacturer's instructionsusing a GENE PULSER® and Pulse Controller at 50 μF, 25 mAmp, 1.8 kV witha 2 mm gap cuvette according to the manufacturer's procedure.

The electroporated cells were incubated in SOC medium with shaking at250 rpm for 2 hours at 28° C. before being plated on the followingselective media: LB medium supplemented with 50 μg of kanamycin per ml;LB medium supplemented with 50 μg of kanamycin per ml and 15 μg ofchloramphencol per ml; and/or LB medium supplemented with 50 μg ofkanamycin per ml, 15 μg of chloramphencol per ml, and 12.5 μg ofampicillin per ml.

From dilution plating of the electroporation onto LB medium supplementedwith kanamycin and chloramphencol medium, it was determined thatapproximately 72,000 colonies were present containing a cDNA libraryplasmid with a SigA2 transposition per electroporation and thatapproximately 69 colonies were recovered under triple selection (LB,kanamycin, chorlamphenicol, ampicillin). Further electroporation andplating experiments were performed until 445 total colonies wererecovered under triple selection. The colonies were miniprepped using aQIAPREP® 96 Turbo Miniprep Kit (QIAGEN GmbH Corporation, Hilden,Germany). Plasmids were sequenced with the transposon forward andreverse primers (primers A and B), shown below, according to theprocedure disclosed in WO 2001/77315 (page 28)

Primer A: (SEQ ID NO: 5) 5′-AGCGTTTGCGGCCGCGATCC-3′ Primer B:(SEQ ID NO: 6) 5′-TTATTCGGTCGAAAAGGATCC-3′

Example 6 Cloning of the Trichophaea saccata Family GH6 Gene into anAspergillus oryzae Expression Vector

The Trichophaea saccata Family GH6 cDNA encoding cellobiohydrolase wassubcloned into the Aspergillus expression vector pMStr57 (WO2004/032648) by PCR amplifying the protein coding sequence from the cDNAlibrary SBL0521, described in Example 3 above, with the two syntheticoligonucleotide primers shown below.

Primer 848: (SEQ ID NO: 7)5′-ACACAACTGGGGATCCTCATCATGAAGAACTTCCTTCTGG-3′ Primer 849:(SEQ ID NO: 8) 5′-CCCTCTAGATCTCGAGTTACGTGAAGCTAGGATTAGCATT-3′

The amplification was performed using IPROOF™ High Fidelity 2× MasterMix (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) following themanufacturer's instructions. The amplification reaction was composed ofSBL0521 pool DNA as template, 25 μmol each of primers 848 and 849, and25 μl of IPROOF™ High Fidelity 2× Master Mix in a final volume of 50 μl.The amplification was performed by pre-denaturing at 98° C. for 2minutes; 5 cycles each with denaturing at 98° C. for 10 seconds,annealing at 65° C. for 10 seconds, and elongation at 72° C. for 1minute; and 25 cycles each with denaturing at 98° C. for 10 seconds, andcombined annealing extension at 72° C. for 1 minute. A final elongationwas made at 72° C. for 10 minutes.

A PCR product of 1.4 kb was separated from residual reaction componentsusing a GFX® PCR DNA and Gel Band Purification Kit (GE Healthcare UKLimited, Buckinghamshire, UK) according to the manufacturer'sinstructions.

The PCR fragment was cloned into Barn HI and Xho I digested pMStr57using an IN-FUSION™ Dry-Down PCR Cloning Kit (Clontech Laboratories,Inc., Mountain View, Calif., USA). Approximately 50 ng of PCR productand 200 ng of vector in a total volume of 10 μl were added to theIN-FUSION™ Dry-Down pellet. The reaction was performed according to themanufacturer's instructions. The Trichophaea saccata Family GH6cellobiohydrolase encoding DNA of the resulting Aspergillus expressionconstruct, pMStr179, was sequenced and the sequence agreed completelywith the cellobiohydrolase coding sequence of SEQ ID NO: 1.

The same PCR fragment was cloned into the pCR®-BluntII-TOPO vector(Invitrogen, Life Technologies, Carlsbad, Calif., USA) using a ZeroBlunt TOPO PCR Cloning Kit, to generate pMStr199. The Trichophaeasaccata Family GH6 cellobiohydrolase encoding DNA of pMStr199 wassequenced and the sequence agreed completely with the cellobiohydrolasecoding sequence of SEQ ID NO: 1. E. coli strain NN059235, containingpMStr199, was deposited with the Deutsche Sammlung von Mikroorganismenand Zellkulturen GmbH (DSM), Braunschweig, Germany, on Feb. 24, 2010 andassigned the accession number DSM 23379.

Example 7 Characterization of the Trichophaea Saccata cDNA Encoding aFamily GH6 Polypeptide Having Cellobiohydrolase Activity

The nucleotide sequence and deduced amino acid sequence of theTrichophaea saccata cellobiohydrolase cDNA are shown in SEQ ID NO: 1 andSEQ ID NO: 2, respectively. The coding sequence is 1344 bp including thestop codon. The encoded predicted protein is 447 amino acids. Using theSignalP program (Nielsen et al., 1997, Protein Engineering 10:1-6), asignal peptide of 16 residues was predicted. The predicted matureprotein contains 431 amino acids with a predicted molecular mass of 45.3kDa and an isoelectric point of 5.06.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofEMBOSS with gap open penalty of 10, gap extension penalty of 0.5, andthe EBLOSUM62 matrix. The alignment showed that the deduced amino acidsequence of the Trichophaea saccata cDNA encoding a Family GH6polypeptide having cellobiohydrolase activity shares 64% identity(excluding gaps) to the deduced amino acid sequence of acellobiohydrolase from Aspergillus fumigatus (GENESEQP:ABB80166).

Example 8 Expression of the Trichophaea saccata Family GH6 PolypeptideHaving Cellobiohydrolase Activity in Aspergillus oryzae BECh2

The Aspergillus oryzae strain BECh2 (WO 2000/39322) was transformed withpMStr179 according to Christensen et al., 1988, Biotechnology 6,1419-1422 and WO 2004/032648. Ten transformants were cultured for 4 daysat 30° C. in 750 μl of DAP2C-1 medium (WO 2004/032648), in which 2%glucose was substituted for maltodextrin. Samples were monitored bySDS-PAGE using a CRITERION™ XT Precast 12% Bis-Tris gel (Bio-RadLaboratories, Inc., Hercules, Calif., USA) according to themanufacturer's instructions. LMW standards from an Amersham LowMolecular Weight Calibration Kit for SDS Electrophoresis (GE HealthcareUK Limited, Buckinghamshire, UK) were used as molecular weight markers.The gel was stained with INSTANTBLUE™ (Expedeon Protein Solutions,Cambridge, UK). Eight transformants produced a novel protein doublet inthe range of 55-60 kDa.

Two of these transformants, designated Aspergillus oryzae MStr335 andMStr336, were isolated twice by dilution streaking conidia on selectivemedium (amdS) containing 0.01% TRITON® X-100 to limit colony size.

Example 9 Large Shake Flask Cultures of the Recombinant Aspergillusoryzae MStr335 for the Production of the Trichophea saccata Family GH6Polypeptide

Spores from four confluent COVE N slants of Aspergillus oryzae MStr335spores were collected with a solution of 0.01% TWEEN® 20 and used toinoculate 21 shake flasks each containing 150 ml of DAP2C-1 medium (WO2004/032648) in which 2% glucose was substituted for maltodextrin. Theflasks were incubated at 30° C. with constant shaking at 200 rpm for 3days. Fungal mycelia and spores were removed at harvesting by firstfiltering the fermentation broth through a sandwich of 3 glassmicrofiber filters with increasing particle retention sizes of 1.6 μm,1.2 μm and 0.7 μm, and then filtering through a 0.45 μm filter.

Example 10 Pretreated Corn Stover Hydrolysis Assay

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using 1.4 wt % sulfuric acid at 165°C. and 107 psi for 8 minutes. The water-insoluble solids in thepretreated corn stover (PCS) contained 56.5% cellulose, 4.6%hemicellulose and 28.4% lignin. Cellulose and hemicellulose weredetermined by a two-stage sulfuric acid hydrolysis with subsequentanalysis of sugars by high performance liquid chromatography using NRELStandard Analytical Procedure #002. Lignin was determinedgravimetrically after hydrolyzing the cellulose and hemicellulosefractions with sulfuric acid using NREL Standard Analytical Procedure#003.

Unmilled, unwashed PCS (whole slurry PCS) was prepared by adjusting thepH of PCS to 5.0 by addition of 10 M NaOH with extensive mixing, andthen autoclaving for 20 minutes at 120° C. The dry weight of the wholeslurry PCS was 29%. Milled unwashed PCS (dry weight 32.35%) was preparedby milling whole slurry PCS in a Cosmos ICMG 40 wet multi-utilitygrinder (EssEmm Corporation, Tamil Nadu, India).

The hydrolysis of PCS was conducted using 2.2 ml deep-well plates(Axygen, Union City, Calif., USA) in a total reaction volume of 1.0 ml.The hydrolysis was performed with 50 mg of insoluble PCS solids per mlof 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfateand various protein loadings of various enzyme compositions (expressedas mg protein per gram of cellulose). Enzyme compositions were preparedand then added simultaneously to all wells in a volume ranging from 50μl to 200 μl, for a final volume of 1 ml in each reaction. The plate wasthen sealed using an ALPS-300™ plate heat sealer (Abgene, Epsom, UnitedKingdom), mixed thoroughly, and incubated at a specific temperature for72 hours. All experiments reported were performed in triplicate.

Following hydrolysis, samples were filtered using a 0.45 μm MULTISCREEN®96-well filter plate (Millipore, Bedford, Mass., USA) and filtratesanalyzed for sugar content as described below. When not usedimmediately, filtered aliquots were frozen at −20° C. The sugarconcentrations of samples diluted in 0.005 M H2SO4 were measured using a4.6×250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules,Calif., USA) by elution with 0.05% w/w benzoic acid-0.005 M H2SO4 at 65°C. at a flow rate of 0.6 ml per minute, and quantitation by integrationof the glucose, cellobiose, and xylose signals from refractive indexdetection (CHEMSTATION®, AGILENT® 1100 HPLC, Agilent Technologies, SantaClara, Calif., USA) calibrated by pure sugar samples. The resultantglucose and cellobiose equivalents were used to calculate the percentageof cellulose conversion for each reaction.

Glucose, cellobiose, and xylose were measured individually. Measuredsugar concentrations were adjusted for the appropriate dilution factor.In case of unwashed PCS, the net concentrations ofenzymatically-produced sugars were determined by adjusting the measuredsugar concentrations for corresponding background sugar concentrationsin unwashed PCS at zero time point. All HPLC data processing wasperformed using MICROSOFT EXCEL™ software (Microsoft, Richland, Wash.,USA).

The degree of cellulose conversion to glucose was calculated using thefollowing equation: % conversion=glucose concentration/glucoseconcentration in a limit digest. To calculate total conversion theglucose and cellobiose values were combined. Cellobiose concentrationwas multiplied by 1.053 in order to convert to glucose equivalents andadded to the glucose concentration. The degree of total celluloseconversion was calculated using the following equation: %conversion=[glucose concentration+1.053× (cellobioseconcentration)]/[(glucose concentration+1.053× (cellobioseconcentration) in a limit digest]. The 1.053 factor for cellobiose takesinto account the increase in mass when cellobiose is converted toglucose. In order to calculate % conversion, a 100% conversion point wasset based on a cellulase control (50-100 mg of Trichoderma reeseicellulase per gram cellulose), and all values were divided by thisnumber and then multiplied by 100. Triplicate data points were averagedand standard deviation was calculated.

Example 11 Preparation of Aspergillus fumigatus NN055679 Cel7ACellobiohydrolase I

A tfasty search (Pearson et al., 1997, Genomics 46:24-36) of theAspergillus fumigatus partial genome sequence (The Institute for GenomicResearch, Rockville, Md.) was performed using as query a Cel7cellobiohydrolase protein sequence from Trichoderma reesei (AccessionNo. P00725). Several genes were identified as putative Family GH7homologs based upon a high degree of similarity to the query sequence atthe amino acid level. One genomic region with significant identity tothe query sequence was chosen for further study, and the correspondinggene was named cel7A.

Two synthetic oligonucleotide primers shown below were designed to PCRamplify an Aspergillus fumigatus NN055679 cel7A cellobiohydrolase I gene(SEQ ID NO: 9 [DNA sequence] and SEQ ID NO: 10 [deduced amino acidsequence]) from genomic DNA of Aspergillus fumigatus prepared asdescribed in WO 2005/047499.

Forward primer: (SEQ ID NO: 11) 5′-gggcATGCTGGCCTCCACCTTCTCC-3′Reverse primer: (SEQ ID NO: 12) 5′-gggttaattaaCTACAGGCACTGAGAGTAA-3′

Upper case letters represent the coding sequence. The remainder of thesequence provides restriction endonuclease sites for Sph I and Pac I inthe forward and reverse sequences, respectively. Using these primers,the Aspergillus fumigatus cel7A gene was amplified using standard PCRmethods and the reaction product isolated by 1% agarose gelelectrophoresis using TAE buffer and purified using a QIAQUICK® GelExtraction Kit (QIAGEN Inc., Valencia, Calif., USA) according to themanufacturer's instructions.

The fragment was digested with Sph I and Pac I and ligated into theexpression vector pAlLo2 also digested with Sph I and Pac I according tostandard procedures. The ligation products were transformed into E. coliXL10 SOLOPACK® cells (Stratagene, La Jolla, Calif., USA) according tothe manufacturer's instructions. An E. coli transformant containing aplasmid of the correct size was detected by restriction digestion andplasmid DNA was prepared using a BIOROBOT® 9600 (QIAGEN Inc., Valencia,Calif., USA). DNA sequencing of the insert gene from this plasmid wasperformed with an Applied Biosystems Model 377 XL Automated DNASequencer (Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif.,USA) using dye-terminator chemistry (Giesecke et al., 1992, Journal ofVirology Methods 38: 47-60) and primer walking strategy. Nucleotidesequence data were scrutinized for quality and all sequences werecompared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA). The nucleotide sequencewas shown to match the genomic sequence determined by TIGR (SEQ ID NO: 9[DNA sequence] and SEQ ID NO: 10 [deduced amino acid sequence]). Theresulting plasmid was named pEJG93.

Aspergillus oryzae JaL250 (WO 99/61651) protoplasts were preparedaccording to the method of Christensen et al., 1988, supra andtransformed with 5 μg of pEJG93 (as well as pAlLo2 as a vector control)was used to transform Aspergillus oryzae JaL250. The transformation ofyielded about 100 transformants. Ten transformants were isolated toindividual PDA plates.

Confluent PDA plates of five of the ten transformants were washed with 5ml of 0.01% TWEEN® 20 and inoculated separately into 25 ml of MDU2BPmedium in 125 ml glass shake flasks and incubated at 34° C., 250 rpm.Five days after incubation, 0.5 μl of supernatant from each culture wasanalyzed using 8-16% Tris-Glycine SDS-PAGE gels (Invitrogen, Carlsbad,Calif., USA) according to the manufacturer's instructions. SDS-PAGEprofiles of the cultures showed that one of the transformants had amajor band of approximately 70 kDa. This transformant was namedAspergillus oryzae JaL250EJG93.

Five hundred ml of shake flask medium were added to a 2800 ml shakeflask. The shake flask medium was composed of 45 g of maltose, 2 g ofK₂HPO₄, 12 g of KH₂PO₄, 1 g of NaCl, 1 g of MgSO₄.7H₂O, 7 g of yeastextract, 2 g of urea, 0.5 ml of trace elements solution, and deionizedwater to 1 liter. The trace elements solution was composed of 13.8 g ofFeSO₄.7H₂O, 14.3 g of ZnSO₄.7H₂O, 8.5 g of MnSO₄.H₂O, 2.5 g ofCuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 3 g of citric acid, and deionized waterto 1 liter. Two shake flasks were inoculated with a suspension of a PDAplate of Aspergillus oryzae JaL250EJG93 with 0.01% TWEEN® 80 andincubated at 34° C. on an orbital shaker at 200 rpm for 120 hours. Thebroth was filtered using a 0.7 μm Whatman glass filter GF/F (Whatman,Piscataway, N.J., USA) followed by a 0.22 μm EXPRESS™ Plus Membrane(Millipore, Bedford, Mass., USA).

Filtered broth was concentrated and buffer exchanged with 20 mM Tris-HClpH 8.5 using a tangential flow concentrator (Pall Filtron, Northborough,Mass., USA) equipped with a 10 kDa polyethersulfone membrane (PallFiltron, Northborough, Mass., USA). Protein concentration was determinedusing a Microplate BCA™ Protein Assay Kit (Thermo Fischer Scientific,Waltham, Mass., USA) in which bovine serum albumin was used as a proteinstandard.

Example 12 Preparation of Thermoascus aurantiacus CGMCC 0670 Cel5AEndoglucanase II

Thermoascus aurantiacus CGMCC 0670 cDNA encoding a Cel5A endoglucanaseII (SEQ ID NO: 13 [DNA sequence] and SEQ ID NO: 14 [deduced amino acidsequence]) was cloned according to the following procedure. The T.aurantiacus strain was grown in 80 ml of CBH1 medium (2.5% AVICEL®, 0.5%glucose, 0.14% (NH₄)₂SO₄) in 500 ml Erlenmeyer baffled flasks at 45° C.for 3 days with shaking at 165 rpm. Mycelia were harvested bycentrifugation at 7000 rpm for 30 minutes and stored at −80 C before usefor RNA extraction. RNA was isolated from 100 mg of mycelia using aRNEASY® Plant Mini Kit (QIAGEN Inc., Valencia, Calif., USA).

The cDNA for the Thermoascus aurantiacus endoglucanase was isolated byRT PCR using a 3′ RACE system and a 5′ RACE system and primers BG025-1,BG025-2, BG025-3, and BG025-4 shown below to the N-terminal amino acids.

Primer BG025-1: (SEQ ID NO: 15)5′-AA(T/C)GA(A/G)TC(T/C/A/G)GG(T/C/A/G)GC(T/C/A/G) GAATT-3′Primer BG025-2: (SEQ ID NO: 16)5′-AA(T/C)GA(A/G)TC(T/C/A/G)GG(T/C/A/G)GC(T/C/A/G) GAGTT-3′Primer BG025-3: (SEQ ID NO: 17)5′-AA(T/C)GA(A/G)AG(T/C)GG(T/C/A/G)GC(T/C/A/G) GAATT-3′ Primer BG025-4:(SEQ ID NO: 18) 5′-AA(T/C)GA(A/G)AG(T/C)GG(T/C/A/G)GC(T/C/A/G) GAGTT-3′

The RT PCR products were ligated into plasmid pGEM-T using a pGEM-TVector System (Promega, Madison, Wis., USA) and transformed into E. colistrain JM109. A single clone harboring a plasmid named pBGC1009containing the endoglucanase cDNA was isolated.

PCR primers were designed to amplify the cDNA encoding the T aurantiacusendoglucanase from plasmid pBGC1009. Restriction enzyme sites Bsp HI andPac I were incorporated for in-frame cloning into Aspergillus oryzaeexpression plasmid pBM120a (WO 2006/039541).

Primer 996261: (SEQ ID NO: 19) 5′-GATCTCATGAAGCTCGGCTCTCTCGT-3′    BspHIPrimer 996167: (SEQ ID NO: 20) 5′-TTAATTAATCAAAGATACGGAGTCAAAATAGG-3′   PacI

The fragment of interest was amplified by PCR using an EXPAND™ HighFidelity PCR System. The PCR amplification reaction mixture contained 1μl of 0.09 μg/μl pBGC1009, 1 μl of primer 996261 (50 μmol/μl), 1 μl ofprimer 996167 (50 μmol/μl), 5 μl of 10×PCR buffer with 15 mM MgCl₂, 1 μlof dNTP mix (10 mM each), 37.25 μl of water, and 0.75 μl (3.5 U/μl) ofDNA polymerase mix. An EPPENDORF® MASTERCYCLER® thermocycler (EppendorfScientific, Inc., Westbury, N.Y., USA) was used to amplify the fragmentprogrammed for 1 cycle at 94° C. for 2 minutes; 10 cycles each at 94° C.for 15 seconds, 55° C. for 30 seconds, 72° C. for 1.5 minutes; 15 cycleseach at 94° C. for 15 seconds, 55° C. for 30 seconds, and 72° C. for 1.5minutes plus 5 second elongation at each successive cycle; 1 cycle at72° C. for 7 minutes; and a 4° C. hold.

The 1008 bp PCR product was purified by 1% agarose gel electrophoresisusing TAE buffer, excised from the gel, and purified using a QIAQUICK®Gel Purification Kit (QIAGEN Inc., Valencia, Calif., USA). The purifiedproduct was ligated directly into pCR®2.1-TOPO® according to themanufacturer's instructions. The resulting plasmid was named pBM124a.

Plasmid pBM124a was digested with Bsp HI and Pac I, purified by 1%agarose gel electrophoresis using TAE buffer, excised from the gel, andpurified using a QIAQUICK® Gel Purification Kit. The plasmid fragmentwas ligated to the vector pBM120a, which was digested with Nco I and PacI. The resulting expression plasmid was designated pBM123a.

Plasmid pBM123a contains a duplicate NA2-TPI promoter driving expressionof the Thermoascus aurantiacus endoglucanase cDNA clone, the AMGterminator, and amdS as a selectable marker.

Aspergillus oryzae BECh2 (WO 2000/139322) protoplasts were preparedaccording to the method of Christensen et al., 1988, supra andtransformed with 6 μg of pBM123a. Primary transformants were selected onCOVE plates for 5 days. Transformants were spore purified twice prior toshake flask analysis.

Spores of the transformants were inoculated into 25 ml of MY25 medium in125 ml shake flasks. The cultures were incubated at 34° C., 200 rpm on aplatform shaker for five days. On day 3 and day 5, culture supernatantswere harvested and clarified by centrifugation to remove mycelia. Twentymicroliters of supernatant from three transformants were analyzed usinga CRITERION® stain-free, 10-20% gradient SDS-PAGE gel (Bio-RadLaboratories, Inc., Hercules, Calif., USA) according to themanufacturer's instructions. SDS-PAGE profiles of the cultures showedthat all transformants had a new major band of approximately 32 kDa. Onetransformant was chosen and named A. oryzae EXP00858.

Plastic, non-baffled 500 ml shake flasks containing 100 ml of SY50medium were inoculated with 0.1 ml of a spore stock of A. oryzaeEXP00858, and incubated at 34° C., 200 rpm for 24 hours to produce aseed culture. Fifty ml of the seed culture was inoculated into a 2 literfermentation tank containing 2 liters of medium composed per liter of0.5 g of pluronic acid, 30 g of sucrose, 2 g of MgSO₄.7H₂O, 2 g ofanhydrous KH₂PO₄, 1 g of citric acid, 2 g of (NH₄)₂SO₄, 1 g of K₂SO₄, 20g of yeast extract, and 0.5 g of 200×AMG trace metals solution, pH 5.0.The fermentation was fed with a maltose feed. The pH was controlledusing 5N H₃PO₄ and 15% NH₄OH and maintained at 5.0 and then raised to5.25. Temperature was maintained 34.0° C.+/−1.0° C. Agitation was 1000rpm. Airflow was 1.0 vvm.

A 200 ml volume of cell-free supernatant was diluted to 1 liter withdeionized water. The pH was adjusted to 8 and the sample filtersterilized using a 0.22 μm polyethersulphone (PES) filter. The filtersterilized sample was loaded onto a 250 ml Q SEPHAROSE™ Fast Flow column(GE Healthcare, Piscataway, N.J., USA) pre-equilibrated with 25 mM TrispH 8. The enzyme was eluted from the column with a 0 to 1 M NaOHgradient in the same buffer. The fractions containing beta-glucosidaseactivity were pooled (400 ml) and the enzyme concentration calculatedfrom the theoretic extinction coefficient and the absorbance of thesample at 280 nm.

Example 13 Preparation of Thermoascus aurantiacus CGMCC 0583 GH61APolypeptide Having Cellulolytic Enhancing Activity

Thermoascus aurantiacus CGMCC 0583 GH61A polypeptide having cellulolyticenhancing activity (SEQ ID NO: 21 [DNA sequence] and SEQ ID NO: 22[deduced amino acid sequence]) was recombinantly prepared according toWO 2005/074656 using Aspergillus oryzae JaL250 as a host. Therecombinantly produced Thermoascus aurantiacus GH61A polypeptide wasfirst concentrated by ultrafiltration using a 10 kDa membrane, bufferexchanged into 20 mM Tris-HCl pH 8.0, and then purified using a 100 ml QSEPHAROSE® Big Beads column (GE Healthcare, Piscataway, N.J., USA) with600 ml of a 0-600 mM NaCl linear gradient in the same buffer. Fractionsof 10 ml were collected and pooled based on SDS-PAGE.

The pooled fractions (90 ml) were then further purified using a 20 mlMONO Q® column (GE Healthcare, Piscataway, N.J., USA) with 500 ml of a0-500 mM NaCl linear gradient in the same buffer. Fractions of 6 ml werecollected and pooled based on SDS-PAGE. The pooled fractions (24 ml)were concentrated by ultrafiltration using a 10 kDa membrane, andchromatographed using a 320 ml SUPERDEX® 75 SEC column (GE Healthcare,Piscataway, N.J., USA) with isocratic elution of approximately 1.3liters of 150 mM NaCl-20 mM Tris-HCl pH 8.0. Fractions of 20 ml werecollected and pooled based on SDS-PAGE. Protein concentration wasdetermined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard.

Example 14 Preparation of Aspergillus fumigatus NN055679 Cel3ABeta-Glucosidase

Aspergillus fumigatus NN055679 Cel3A beta-glucosidase (SEQ ID NO: 23[DNA sequence] and SEQ ID NO: 24 [deduced amino acid sequence]) wasrecombinantly prepared according to WO 2005/047499 using Trichodermareesei RutC30 as a host.

Filtered broth was concentrated and buffer exchanged using a tangentialflow concentrator equipped with a 10 kDa polyethersulfone membrane with20 mM Tris-HCl pH 8.5. The sample was loaded onto a Q SEPHAROSE® HighPerformance column (GE Healthcare, Piscataway, N.J., USA) equilibratedin 20 mM Tris pH 8.5, and bound proteins were eluted with a lineargradient from 0-600 mM sodium chloride. The fractions were concentratedand loaded onto a SUPERDEX® 75 HR 26/60 column GE Healthcare,Piscataway, N.J., USA) equilibrated with 20 mM Tris-150 mM sodiumchloride pH 8.5. Protein concentration was determined using a MicroplateBCA™ Protein Assay Kit in which bovine serum albumin was used as aprotein standard.

Example 15 Preparation of Aspergillus fumigatus NN055679 GH10 Xylanase

Aspergillus fumigatus NN055679 GH10 xylanase (xyn3) (SEQ ID NO: 25 [DNAsequence] and SEQ ID NO: 26 [deduced amino acid sequence]) was preparedrecombinantly according to WO 2006/078256 using Aspergillus oryzae BECh2as a host.

The filtered broth was desalted and buffer-exchanged into 20 mM Tris−150 mM NaCl pH 8.5 using a HIPREP® 26/10 Desalting Column (GEHealthcare, Piscataway, N.J., USA) according to the manufacturer'sinstructions. Protein concentration was determined using a MicroplateBCA™ Protein Assay Kit with bovine serum albumin as a protein standard.

Example 16 Evaluation of Trichophaea saccata Family GH6Cellobiohydrolase in a High-Temperature Enzyme Composition Using MilledUnwashed PCS at 50-65° C.

The Trichophaea saccata Family GH6 cellobiohydrolase was evaluated in ahigh-temperature enzyme composition at 50° C., 55° C., 60° C., and 65°C. using milled unwashed PCS as a substrate. The high-temperature enzymecomposition included 35% Aspergillus fumigatus Cel7A CBHI, 25%Trichophaea saccata Family GH6 polypeptide CBHII, 15% Thermoascusaurantiacus Cel5A EGII, 15% Thermoascus aurantiacus GH61A polypeptidehaving cellulolytic enhancing activity, 5% Aspergillus fumigatus Cel3Abeta-glucosidase, and 5% Aspergillus fumigatus GH10 xyn3 xylanase. Thehigh-temperature enzyme composition was added to PCS hydrolysisreactions at 3.0 mg total protein per g cellulose, and the hydrolysisresults were compared with the results for a similar high-temperatureenzyme composition without added GH6 polypeptide (2.25 mg protein per gcellulose).

The assay was performed as described in Example 10. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

The results shown in FIG. 1 demonstrated that at 50° C. and 55° C., thehigh-temperature enzyme composition that included 25% Trichophaeasaccata Family GH6 cellobiohydrolase significantly outperformed theenzyme composition containing no GH6 cellobiohydrolase. However, at 60°C. and 65° C., the inclusion of Trichophaea saccata Family GH6cellobiohydrolase in a high-temperature enzyme composition provided onlya small improvement over the performance of the enzyme compositioncontaining no GH6 cellobiohydrolase.

DEPOSIT OF BIOLOGICAL MATERIAL

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Mascheroder Weg 1 B, D-38124 Braunschweig,Germany, and given the following accession number:

Deposit Accession Number Date of Deposit E. coli NN059235 DSM 23379 Feb.24, 2010

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by foreign patent laws to be entitledthereto. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The present invention is further described by the following numberedparagraphs:

[1] An isolated polypeptide having cellobiohydrolase activity, selectedfrom the group consisting of: (a) a polypeptide comprising an amino acidsequence having at least 65% identity to the mature polypeptide of SEQID NO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizesunder at least medium stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the genomic DNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 1, or(iii) a full-length complementary strand of (i) or (ii); (c) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 65% identity to the mature polypeptide coding sequenceof SEQ ID NO: 1; and (d) a variant comprising a substitution, deletion,and/or insertion of one or more (several) amino acids of the maturepolypeptide of SEQ ID NO: 2.

[2] The polypeptide of paragraph 1, comprising an amino acid sequencehaving at least 65% identity to the mature polypeptide of SEQ ID NO: 2.

[3] The polypeptide of paragraph 2, comprising an amino acid sequencehaving at least 70% identity to the mature polypeptide of SEQ ID NO: 2.

[4] The polypeptide of paragraph 3, comprising an amino acid sequencehaving at least 75% identity to the mature polypeptide of SEQ ID NO: 2.

[5] The polypeptide of paragraph 4, comprising an amino acid sequencehaving at least 80% identity to the mature polypeptide of SEQ ID NO: 2.

[6] The polypeptide of paragraph 5, comprising an amino acid sequencehaving at least 85% identity to the mature polypeptide of SEQ ID NO: 2.

[7] The polypeptide of paragraph 6, comprising an amino acid sequencehaving at least 90% identity to the mature polypeptide of SEQ ID NO: 2.

[8] The polypeptide of paragraph 7, comprising an amino acid sequencehaving at least 95% identity to the mature polypeptide of SEQ ID NO: 2.

[9] The polypeptide of paragraph 8, comprising an amino acid sequencehaving at least 97% identity to the mature polypeptide of SEQ ID NO: 2.

[10] The polypeptide of paragraph 1, comprising or consisting of theamino acid sequence of SEQ ID NO: 2; or a fragment thereof havingcellobiohydrolase activity.

[11] The polypeptide of paragraph 10, comprising or consisting of theamino acid sequence of SEQ ID NO: 2.

[12] The polypeptide of paragraph 10, comprising or consisting of themature polypeptide of SEQ ID NO: 2.

[13] The polypeptide of paragraph 1, which is encoded by apolynucleotide that hybridizes under at least medium stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of(i) or (ii).

[14] The polypeptide of paragraph 13, which is encoded by apolynucleotide that hybridizes under at least medium-high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of(i) or (ii).

[15] The polypeptide of paragraph 14, which is encoded by apolynucleotide that hybridizes under at least high stringency conditionswith (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii)the genomic DNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or(ii).

[16] The polypeptide of paragraph 15, which is encoded by apolynucleotide that hybridizes under at least very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of(i) or (ii).

[17] The polypeptide of paragraph 1, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 65%identity to the mature polypeptide coding sequence of SEQ ID NO: 1

[18] The polypeptide of paragraph 17, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 70%identity to the mature polypeptide coding sequence of SEQ ID NO: 1

[19] The polypeptide of paragraph 18, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 75%identity to the mature polypeptide coding sequence of SEQ ID NO: 1

[20] The polypeptide of paragraph 19, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 80%identity to the mature polypeptide coding sequence of SEQ ID NO: 1

[21] The polypeptide of paragraph 20, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 85%identity to the mature polypeptide coding sequence of SEQ ID NO: 1

[22] The polypeptide of paragraph 21, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 90%identity to the mature polypeptide coding sequence of SEQ ID NO: 1

[23] The polypeptide of paragraph 22, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 95%identity to the mature polypeptide coding sequence of SEQ ID NO: 1

[24] The polypeptide of paragraph 23, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 97%identity to the mature polypeptide coding sequence of SEQ ID NO: 1

[25] The polypeptide of paragraph 1, which is encoded by apolynucleotide comprising or consisting of the nucleotide sequence ofSEQ ID NO: 1; or a subsequence thereof encoding a fragment havingcellobiohydrolase activity.

[26] The polypeptide of paragraph 25, which is encoded by apolynucleotide comprising or consisting of the nucleotide sequence ofSEQ ID NO: 1.

[27] The polypeptide of paragraph 25, which is encoded by apolynucleotide comprising or consisting of the mature polypeptide codingsequence of SEQ ID NO: 1.

[28] The polypeptide of paragraph 1, wherein the polypeptide is avariant comprising a substitution, deletion, and/or insertion of one ormore (several) amino acids of the mature polypeptide of SEQ ID NO: 2.

[29] The polypeptide of paragraph 1, which is encoded by thepolynucleotide contained in plasmid pMStr199 which is contained in E.coli DSM 23379.

[30] The polypeptide of any of paragraphs 1-29, wherein the maturepolypeptide is amino acids 17 to 447 of SEQ ID NO: 2.

[31] The polypeptide of any of paragraphs 1-30, wherein the maturepolypeptide coding sequence is nucleotides 109 to 1401 of SEQ ID NO: 1.

[32] An isolated polynucleotide comprising a nucleotide sequence thatencodes the polypeptide of any of paragraphs 1-31.

[33] A nucleic acid construct comprising the polynucleotide of paragraph32 operably linked to one or more (several) control sequences thatdirect the production of the polypeptide in an expression host.

[34] A recombinant expression vector comprising the polynucleotide ofparagraph 32.

[35] A recombinant host cell comprising the polynucleotide of paragraph32 operably linked to one or more (several) control sequences thatdirect the production of a polypeptide having cellobiohydrolaseactivity.

[36] A method of producing the polypeptide of any of paragraphs 1-31,comprising: (a) cultivating a cell, which in its wild-type form producesthe polypeptide, under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.

[37] A method of producing the polypeptide of any of paragraphs 1-31,comprising: (a) cultivating a host cell comprising a nucleic acidconstruct comprising a nucleotide sequence encoding the polypeptideunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

[38] A method of producing a mutant of a parent cell, comprisingdisrupting or deleting a polynucleotide encoding the polypeptide, or aportion thereof, of any of paragraphs 1-31, which results in the mutantproducing less of the polypeptide than the parent cell.

[39] A mutant cell produced by the method of paragraph 38.

[40] The mutant cell of paragraph 39, further comprising a gene encodinga native or heterologous protein.

[41] A method of producing a protein, comprising: (a) cultivating themutant cell of paragraph 39 or 40 under conditions conducive forproduction of the protein; and (b) recovering the protein.

[42] A method of producing the polypeptide of any of paragraphs 1-31,comprising: (a) cultivating a transgenic plant or a plant cellcomprising a polynucleotide encoding the polypeptide under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.

[43] A transgenic plant, plant part or plant cell transformed with apolynucleotide encoding the polypeptide of any of paragraphs 1-31.

[44] A double-stranded inhibitory RNA (dsRNA) molecule comprising asubsequence of the polynucleotide of paragraph 32, wherein optionallythe dsRNA is a siRNA or a miRNA molecule.

[45] The double-stranded inhibitory RNA (dsRNA) molecule of paragraph44, which is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or moreduplex nucleotides in length.

[46] A method of inhibiting the expression of a polypeptide havingcellobiohydrolase activity in a cell, comprising administering to thecell or expressing in the cell the double-stranded inhibitory RNAmolecule of paragraph 44 or 45.

[47] An isolated polynucleotide encoding a signal peptide comprising orconsisting of amino acids 1 to 16 of SEQ ID NO: 2.

[48] A nucleic acid construct comprising a gene encoding a proteinoperably linked to the polynucleotide of paragraph 47, wherein the geneis foreign to the polynucleotide.

[49] A recombinant expression vector comprising the polynucleotide ofparagraph 47.

[50] A recombinant host cell comprising the polynucleotide of paragraph47 operably linked to a gene encoding a protein, wherein the gene isforeign to the polynucleotide.

[51] A method of producing a protein, comprising: (a) cultivating arecombinant host cell comprising a gene encoding a protein operablylinked to the polynucleotide of paragraph 47, wherein the gene isforeign to the polynucleotide under conditions conducive for productionof the protein; and (b) recovering the protein.

[52] A composition comprising the polypeptide of any of paragraphs 1-31.

[53] A method for degrading or converting a cellulosic material,comprising: treating the cellulosic material with an enzyme compositionin the presence of the polypeptide of any of paragraphs 1-31.

[54] The method of paragraph 53, wherein the cellulosic material ispretreated.

[55] The method of paragraph 53 or 54, further comprising recovering thedegraded cellulosic material.

[56] The method of any of paragraphs 53-55, wherein the enzymecomposition comprises one or more (several) enzymes selected from thegroup consisting of a cellulase, a hemicellulase, an expansin, anesterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.

[57] The method of paragraph 56, wherein the cellulase is one or more(several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[58] The method of paragraph 56, wherein the hemicellulase is one ormore (several) enzymes selected from the group consisting of a xylanase,an acetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[59] The method of any of paragraphs 53-58, wherein the degradedcellulosic material is a sugar.

[60] The method of paragraph 59, wherein the sugar is selected from thegroup consisting of glucose, xylose, mannose, galactose, and arabinose.

[61] A method for producing a fermentation product, comprising: (a)saccharifying a cellulosic material with an enzyme composition in thepresence of the polypeptide of any of paragraphs 1-31; (b) fermentingthe saccharified cellulosic material with one or more (several)fermenting microorganisms to produce the fermentation product; and (c)recovering the fermentation product from the fermentation.

[62] The method of paragraph 61, wherein the cellulosic material ispretreated.

[63] The method of paragraph 61 or 62, wherein the enzyme compositioncomprises one or more (several) enzymes selected from the groupconsisting of a cellulase, a hemicellulase, an expansin, an esterase, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.

[64] The method of paragraph 63, wherein the cellulase is one or more(several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[65] The method of paragraph 63, wherein the hemicellulase is one ormore (several) enzymes selected from the group consisting of a xylanase,an acetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[66] The method of any of paragraphs 61-65, wherein steps (a) and (b)are performed simultaneously in a simultaneous saccharification andfermentation.

[67] The method of any of paragraphs 61-66, wherein the fermentationproduct is an alcohol, an organic acid, a ketone, an amino acid, or agas.

[68] A method of fermenting a cellulosic material, comprising:fermenting the cellulosic material with one or more (several) fermentingmicroorganisms, wherein the cellulosic material is saccharified with anenzyme composition in the presence of the polypeptide of any ofparagraphs 1-31.

[69] The method of paragraph 68, wherein the cellulosic material ispretreated before saccharification.

[70] The method of paragraph 68 or 69, wherein the enzyme compositioncomprises one or more (several) enzymes selected from the groupconsisting of a cellulase, a hemicellulase, an expansin, an esterase, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.

[71] The method of paragraph 70, wherein the cellulase is one or more(several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[72] The method of paragraph 70, wherein the hemicellulase is one ormore (several) enzymes selected from the group consisting of a xylanase,an acetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[73] The method of any of paragraphs 68-72, wherein the fermenting ofthe cellulosic material produces a fermentation product.

[74] The method of paragraph 73, further comprising recovering thefermentation product from the fermentation.

[75] The method of any of paragraphs 73 or 74, wherein the fermentationproduct is an alcohol, an organic acid, a ketone, an amino acid, or agas.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

What is claimed is:
 1. A method for degrading or converting a cellulosicmaterial, comprising: treating the cellulosic material with apolypeptide having cellobiohydrolase activity, wherein the polypeptidecomprises an amino acid sequence having at least 95% sequence identityto amino acids 17 to 447 of SEQ ID NO:
 2. 2. The method of claim 1,further comprising recovering the degraded cellulosic material.
 3. Anucleic acid construct comprising a polynucleotide encoding apolypeptide having cellobiohydrolase activity, wherein thepolynucleotide is operably linked to one or more heterologous controlsequences that direct the production of the polypeptide in an expressionhost, and wherein the polypeptide having cellobiohydrolase activitycomprises an amino acid sequence having at least 95% sequence identityto amino acids 17 to 446 of SEQ ID NO:
 2. 4. A recombinant expressionvector comprising the nucleic acid construct of claim
 3. 5. Arecombinant host cell comprising the nucleic acid construct of claim 3.6. A transgenic plant, plant part or plant cell transformed with thenucleic acid construct of claim
 3. 7. The nucleic acid construct ofclaim 3, wherein the polypeptide having cellobiohydrolase activitycomprises an amino acid sequence having at least 96% sequence identityto amino acids 17 to 447 of SEQ ID NO:
 2. 8. The nucleic acid constructof claim 3, wherein the polypeptide having cellobiohydrolase activitycomprises an amino acid sequence having at least 97% sequence identityto amino acids 17 to 447 of SEQ ID NO:
 2. 9. The nucleic acid constructof claim 3, wherein the polypeptide having cellobiohydrolase activitycomprises an amino acid sequence having at least 98% sequence identityto amino acids 17 to 447 of SEQ ID NO:
 2. 10. The nucleic acid constructof claim 3, wherein the polypeptide having cellobiohydrolase activitycomprises an amino acid sequence having at least 99% sequence identityto amino acids 17 to 447 of SEQ ID NO:
 2. 11. The nucleic acid constructof claim 3, wherein the polypeptide having cellobiohydrolase activitycomprises amino acids 17 to 447 of SEQ ID NO:
 2. 12. The nucleic acidconstruct of claim 3, wherein the polypeptide having cellobiohydrolaseactivity comprises the amino acid sequence of SEQ ID NO:
 2. 13. Thenucleic acid construct of claim 3, wherein the polypeptide havingcellobiohydrolase activity is encoded by a polynucleotide comprising anucleic acid sequence having at least 96% sequence identity tonucleotides 109 to 1401 of SEQ ID NO:
 1. 14. The nucleic acid constructof claim 13, wherein the polypeptide having cellobiohydrolase activityis encoded by a polynucleotide comprising a nucleic acid sequence havingat least 97% sequence identity to nucleotides 109 to 1401 of SEQ IDNO:
 1. 15. The nucleic acid construct of claim 3, wherein thepolypeptide having cellobiohydrolase activity is encoded by apolynucleotide comprising a nucleic acid sequence having at least 98%sequence identity to nucleotides 109 to 1401 of SEQ ID NO:
 1. 16. Thenucleic acid construct of claim 3, wherein the polypeptide havingcellobiohydrolase activity is encoded by a polynucleotide comprising anucleic acid sequence having at least 99% sequence identity tonucleotides 109 to 1401 of SEQ ID NO:
 1. 17. The nucleic acid constructof claim 3, wherein the polypeptide having cellobiohydrolase activity isencoded by a polynucleotide comprising nucleotides 109 to 1401 of SEQ IDNO:
 1. 18. The nucleic acid construct of claim 3, wherein thepolypeptide having cellobiohydrolase activity is encoded by apolynucleotide comprising the nucleotide sequence of SEQ ID NO:
 1. 19.The nucleic acid construct of claim 3, wherein the polypeptide havingcellobiohydrolase activity is encoded by a polynucleotide contained inplasmid pMStr199 which is contained in E. coli DSM
 23379. 20. Thenucleic acid construct of claim 3, wherein the polypeptide havingcellobiohydrolase activity is a variant comprising a substitution,deletion and/or insertion of one or more amino acids of amino acids 17to 447 of SEQ ID NO:
 2. 21. A method of producing a polypeptide havingcellobiohydrolase activity, comprising: (a) cultivating the recombinanthost cell of claim 5 under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.
 22. A method ofproducing a polypeptide having cellobiohydrolase activity, comprising:(a) cultivating the transgenic plant or the plant cell of claim 6 underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.
 23. The method of claim 1, wherein thepolypeptide having cellobiohydrolase activity has at least 97% sequenceidentity to amino acids 17 to 447 of SEQ ID NO:
 2. 24. The method ofclaim 1, wherein the polypeptide having cellobiohydrolase comprisesamino acids 17 to 447 of SEQ ID NO:
 2. 25. The method of claim 1,wherein the polypeptide having cellobiohydrolase activity is a variantcomprising a substitution, deletion and/or insertion of one or moreamino acids of amino acids 17 to 447 of SEQ ID NO: 2.